Immunological process and constructs for increasing the hdl cholesterol concentration by dna vaccination

ABSTRACT

A process for inducing the production of antibodies that bind to cholesteryl ester transfer protein (CETP) is disclosed. That process comprises the steps of: (a) immunizing a mammal with an inoculum containing a recombinant DNA molecule that comprises a DNA sequence that contains (i) a sequence encoding a CETP immunogen that is linked to (ii) a promoter sequence that controls expression of the CETP immunogen, the recombinant DNA molecule being dissolved or dispersed in a vehicle; and (b) maintaining the immunized mammal for a time period sufficient to induce the production of antibodies that bind to CETP, and preferably lessen the transfer of cholesteryl esters from HDL where the blood of the mammal itself contains CETP. Immunogens, inocula, DNA segments, and recombinant DNA molecule vectors useful for carrying out the invention are also disclosed.

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This is a continuation-in-part of application Ser. Nos.08/785,997 and 08/788,882, both filed Jan. 21, 1997.

TECHNICAL FIELD

[0002] The present invention relates to a process for inducingantibodies in a mammal by immunization with DNA that encodes cholesterylester transfer protein (CETP) or a portion thereof, and moreparticularly to an immunological process for amelioratingdyslipoproteinemias in immunized mammals characterized by low HDL/LDLcholesterol ratios by means of those induced antibodies as well asspecific DNA constructs for use in that process.

BACKGROUND OF THE INVENTION

[0003] Cholesteryl ester transfer protein (CETP) is an acidic plasmaglycoprotein that plays a critical role in establishing high densitylipoprotein (HDL), low density lipoprotein (LDL), and very low densitylipoprotein (VLDL) cholesterol blood plasma levels and lipid compositionin plasma [L. Lagrost, Biochem. Biophys. Acta., 1215:209-236 (1994)].Several studies, some of which are discussed below, have demonstratedthat CETP mediates the transfer of cholesteryl esters (CE) from HDLparticles to LDL and VLDL particles, as well as mediating the transferof triglycerides (TG) from LDL and VLDL to HDL particles. Thisreciprocal exchange of CE and TG is the primary means of providing CE toLDL and VLDL particles in many mammalian species. CETP thus mediates thebalanced exchange of cholesteryl esters (CE) and triglycerides (TG)between pro-atherogenic (LDL and VLDL) and anti-atherogenic (HDL)lipoprotein fractions.

[0004] Mammalian species whose blood plasma contains CETP such as humansand other primates, rabbits, and hamsters suffer atherosclerosis andheart disease when exposed to diets rich in cholesterol. Other animalspecies such as mice, rats and dogs lack plasma CETP (measured astransfer activity) and are not susceptible to dietarycholesterol-induced atherosclerosis.

[0005] That CETP contributes to the pathogenesis of atherosclerosis inhumans has been strongly supported by transgenic mouse studies [G.Melchior et al., Trends in Card. Med, 5:83-87 (1995)]. For example,transgenic mice having a mini gene of cynomolgus monkey CETP cDNA plusthe proximal region of the CETP promoter show dietary cholesterolregulation of CETP levels similar to those seen in humans, hamsters andmonkeys. Those transgenic mice expressing high levels of the monkey CETP(levels comparable to human dyslipidemias) exhibit: increased LDL+VLDLcholesterol and apo-B and, decreased HDL cholesterol, LDL-receptor andHMG-CoA reductase mRNA. Atheroma could be induced by high fat diet intransgenic mice with the cynomolgus monkey CETP transgene.

[0006] The CETP amino acid residue and nucleotide sequences of mammalianspecies have been characterized. For example, the human CETP DNAsequence of SEQ ID NO:1 has been determined [D. Drayna et al., Nature,327:632-634 (1987)]. The rabbit CETP DNA sequence of SEQ ID NO:27 hasalso been characterized [M. Nagashima et al., J. Lipid Res.,29:1643-1469 (1988); Kotake et al., J. Lipid Res., 37:599-605(1996) andKotake et al., Biochim. Biophys. Acta., 1347:69-74 (1997)], as has thecynomolgus monkey CETP sequence SEQ ID NO:31 [M. E. Pape et al.,Atherosclerosis and Thrombosis, 11:1759-1771 (1991)]. The human CETPprotein is 476 amino acid residues long, whereas the rabbit CETP proteinis 496 amino acid residues long, and the cynomolgus monkey sequencecontains 476 residues.

[0007] CETP may be a key factor for the global regulation ofatherogenicity of plasma lipoproteins in patients with atherosclerosisor coronary artery disease (CAD). CAD is the number one cause ofmorbidity and mortality in western society. Patients at increased riskfor developing coronary artery disease typically exhibit an enhancedlevel of CETP activity. It has also been reported that CETP has higheraffinity for oxidized LDL than native LDL molecules [L. Lagrost,Biochem. Biophys. Acta., 1215:209-236 (1994)]. High levels of LDLcholesterol (<180 mg/dl) [J. Am. Med. Assoc., 269:3015-3023 (1993) andA. L. Gould et al., Circulation, 91:2274-2282 (1995)]; and low levels ofHDL cholesterol (>35 mg/dl) [G. Assman et al., Excerpta Medica, 46-59(1989) and V. Manninen et al., Circulation, 85:37-45 (1992)] have beenreported to be important contributors to the development ofatherosclerosis.

[0008] Individuals who possess genetic deficiencies of the CETP proteinhave elevated HDL cholesterol levels. Heterozygotes have HDL levels15-20 percent above non-affected controls. It has been suggested thatthere is a 2-3 percent decrease in incidence for CAD for each 1 mg/dlincrease in HDL cholesterol after correction for other risk factors [D.J. Grodon et al., Nature, 79:8-15, (1989)].

[0009] In an experimental model of CETP deficiency in hamsters, it hasbeen shown that passive transfer of mouse anti-human CETP monoclonalantibodies (1C4) inhibited hamster plasma CETP CE transfer by 70-80percent at all times up to 24 hours following injection of 500 μg of 1C4(approximately 3.7 mg/kg body weight). That inhibition of hamsterCETP-mediated transfer in vivo increased hamster HDL cholesterol by 33percent, increased HDL-CE by 31 percent and decreased HDL-TG by 42percent. These results indicate an example of mammalian (hamster)CETP-mediated CE-TG exchange being disrupted by xenogeneic anti-humanCETP monoclonal antibodies, and further demonstrate the use of hamstersas a pre-clinical model for testing CETP inhibition [B. J. Gaynor etal., Atherosclerosis, 110(1):101-109 (1994)].

[0010] In another study reported by G. W. Melchior et al., J. Biol.Chem., 270(36):21068-74 (1995) cynomolgus monkey CETP was shown to havetwo neutral lipid binding sites. A monoclonal antibody to purifiedcynomolgus monkey CETP identified as CMTP-2 was capable of severelyinhibiting triglyceride (TG) transfer, but had a variable effect oncholesteryl ester (CE) transfer.

[0011] Thus, when the monoclonal antibody was administeredsub-cutaneously to cynomolgus monkeys at a dose that inhibited TGtransfer in the plasma by more than 90 percent, there was no detectableeffect on the high density lipoprotein cholesterol level, but the HDL-TGlevels decreased from 13 to 1 mol/mol of HDL. A Fab antibody fragmenthad no effect on CE transfer, but completely blocked TG transfer.Another type of inhibitor, 6-chloromercuric cholesterol, severelyinhibited CE transfer with minimal inhibition of TG transfer. When boththe inhibitory monoclonal antibody and the 6-chloromercuric cholesterolwere added to the assay, both CE and TG transfer were inhibited,indicating that the inhibitors did not compete for the same binding siteon CETP. This study indicated that in vivo administration of xenogeneicmonoclonal antibodies uncoupled CE and TG transfer.

[0012] The inhibitory effects of anti-sense RNA on expression of CETPprotein were reported using vaccinia virus as an expression system. [M.H. Lee et al., J. Biochem. Mol. Biol., 28(3) :243-248 (1995)]. The cDNAfrom CETP was inserted into a transfer vector (pSC11) in sense andanti-sense orientations and then used to construct recombinant vacciniaviruses. Decreased expression of the exogenous CETP cDNA in mouse cellswas clearly evident in the Northern and Western blot analyses as thedose of anti-sense expression increased. Also, in the CETP assay, theCETP activity was decreased compared to the activity obtained from thecell extracts infected with sense constructs only.

[0013] More recently, Sugano et al., J. Biol. Chem., 271(32):19080-19083 (1996) reported upon the in vivo effects of anti-sense CETPRNA administration to rabbits. In that report, decreases in totalcholesterol and CETP activity levels were found 24, 48 and 96 hoursfollowing anti-sense CETP administration, as was an increase in plasmaHDL cholesterol at 48 hours.

[0014] Other methods of inhibition of CETP-mediated transfer aredescribed in the literature. For example, data from Parke-Davis companyhas shown that infusion of 10 to 20 mpk of the small molecule compoundreferred to as PD 140195 into rabbits inhibited CETP activity within 30minutes (measured in an ex vivo assay) [C. Bisgaier et al., Lipids,811-818 (1994)]. Schering-Plough Company has published on the isolationof Wiedendiol-A and -B from a marine sponge and has shown that thisclass of compounds to be low μM inhibitors of CETP-mediated CE transferin vitro [S. Coval et al., Bioorganic & Med. Claim. Lett., 5:605-619(1995)].

[0015] Currently, nicotinic acid and the fibrate drugs are the onlysmall molecule drug therapies that cause significant rises in HDLcholesterol. These drugs are poorly tolerated and must be taken daily.Therapeutic doses of these drugs lead to 15-20 percent increases in HDLcholesterol.

[0016] Three mouse monoclonal antibodies to human CETP that recognize asimilar epitope on CETP, caused parallel and complete in vitroimmunotitration of human plasma CE and triglyceride transfer activities,but only partial inhibition of phospholipid transfer activity [C. B.Hesler et al., J. Biol. Chem., 263(11):5020-5023 (1988)]. Those threemonoclonals were originally designated 5C7, 2H4 and 7E1, but in morerecent publications of the authors, those monoclonals are referred to asTP2, TP1 and TP3, respectively.

[0017] Monoclonal antibody TP2 is directed against an epitope within thelast 26 amino acids of CETP (SEQ ID NO:29) [T. L. Swenson et al., J.Biol. Chem., 264:14318-14326 (1989)], and more particularly to anepitope between about positions 465 and 475 of SEQ ID NO:28 [Tall, J.Lipid Res., 34:1255-1274 (1993)] That monoclonal has been shown to blockCETP-mediated lipid transfer by limiting access to lipid-binding sitesin the carboxy-terminus of CETP.

[0018] In an in vivo study using the xenogeneic mouse monoclonalantibodies (TP1) to CETP, rabbits were intravenously injected with TP1,or irrelevant monoclonal antibodies or saline (control), resulting in aninitial 70 percent inhibition of CETP-mediated CE transfer activity.Inhibition was 45 percent after 48 hours for the TP1-injected animals.HDL-CE increased in TP1-treated animals and reached levels that doubledover initial and control values at 48 hours. HDL-TG fell reciprocally,but HDL protein did not change, suggesting a CE for TG exchange. VLDLCE-TG ratio also decreased. CETP inhibition delayed the initialclearance of radioactively-tagged HDL, suggesting that CETP plays aquantitative role in HDL-CE catabolism in the rabbit, promoting theexchange of TG for CE, and the clearance of CE from plasma [M. E.Whitlock et al., J. Clin. Invest., 84:129-137 (1989)].

[0019] In further animal studies with hamsters, a single sub-cutaneousinjection of TP2 monoclonal antibodies in another illustration ofpassive administration of xenogeneic antibodies decreased CETP-mediatedactivity by 58 percent, lowered LDL+VLDL cholesterol 32 percent andraised HDL cholesterol 24 percent [G. Evans et al., J. Lipid Res.,35:1634-1645 (1994) and S. Zuckerman et al., Lipids, 30:307-311 (1995)].The effect of the TP2 monoclonal antibodies on CETP-mediated CE transferinhibition was evident within 24 hours after injection and was maximizedby 4 days. Lipoproteins returned to control levels 14 days after TP2administration. The shift in the ratio of VLDL+LDL cholesterol to HDLcholesterol levels due to TP2 monoclonal antibody administration wasmore significant in hypercholesterolemic hamsters.

[0020] TP2 also has a higher efficacy in hamsters fed with a westerndiet enriched in cholesterol. CETP-mediated activity was reportedlyincreased in these animals 2-fold over chow-fed hamsters.

[0021] The preparation of recombinant CETP molecules has been reportedby several research groups. For example, in a study reported recently,glutathione S-transferase-human CETP fusion protein (86 kDa) wasexpressed using vaccinia viral transfer vectors transfected into CV-1monkey kidney cells. Using a Western blot assay, the fusion protein wasidentified by polyclonal antibodies against the carboxy-terminal activeregion of CETP fused with GST. After cleavage of the GST portion of thefusion protein, the purified CETP showed biological activity in a CETPin vitro assay [W. H. Yoon et al., Mol. Cells, 5(2):107-113 (1995)] andM. K. Jang et al., J. Biochem. Mol. Biol., 28(3):216-220 (1995)].

[0022] It has also been reported that specific rabbit polyclonalantibodies were produced by immunization with a GST-CETP fusion protein.A full-length CETP cDNA clone isolated from a human heart λgt11 librarywas used to provide the C-terminal 94 bp of CETP after a full lengthCETP molecule expressed in E. coli was found to be insoluble. The λgt11cDNA library was subcloned into pGEX plasmid and a GST-CETP fusionprotein was expressed in E. coli. The CETP-GST fusion protein waspurified by glutathione-Sepharose-4B affinity chromatography and used asan antigen for the production of rabbit polyclonal antibodies. Theantibodies showed good titers, not only against the GST-CETP fusionprotein, but also against a mixture of synthetic peptides correspondingin sequence to two 16-mers from the carboxy-terminal region of humanCETP. The antibodies were said to be useful as an immunological tool fora CETP assay [N. W. Jeong et al., Mol. Cells, 4(4):529-533 (1994)].

[0023] To date there are no published reports on the long-terminhibition of CETP-mediated CE transfer. Passive immunization with theuse of xenogeneic antibodies can only be utilized for a short-termperiod of time because host animals develop antibodies to the xenogeneicimmunoglobulin.

[0024] One strategy for the generation of a host-induced immune responseas compared to xenogeneic antibody use is based on DNA vaccine or“genetic immunization” technology. [W. M. McDonnell et al., N. Engl. J.Med., 334(1): 42045 (1996).] Typically, a DNA vaccine contains a vectorthat includes one or more genes encoding an antigenic portion of avirus, such as an envelope, surface or a core protein. Host cells takeup the DNA vector, express the heterologous gene, and produce thecorresponding viral protein inside the cell.

[0025] One advantage of this approach is that the viral protein entersand is processed by the cell's major histocompatibility complex (MHC)class I pathway. MHC class I molecules carry peptide fragments to thecell surface where they evoke cell-mediated immunity by stimulating CD⁺8cytotoxic T cells. Standard vaccine antigens enter cells by phagocytosisor endocytosis and are processed through the MHC class II system, tostimulate antibody responses.

[0026] The rapid development of DNA vaccine technology within the pastseveral years was spurred by the report that direct injections of a genefrom the influenza A virus could be used to immunize mice against thedisease. [J. B. Ulmer et al., Science, 259: 1745-1749 (1993).] Sincethen, induction of antibodies has been reported for a variety ofpathogen-derived proteins including the influenza NP, HA, M1 proteins;HIV Env, Gag, Rev proteins; bovine herpes virus gp; hepatitis B virussurface and core antigens; rabies virus gp, NP, Plasmodium sp. CSP;Leishmania major gp63; Mycobacterium tuberculosis HSP65, Ag85; HepatitisC virus nucleocapsid protein; Herpes simplex virus gB, gD, ICP27;Papillomavirus L1; Human T-cell leukemia virus type 1 Env; Lymphocyticchoriomeningitis virus NP; Bacillus thuringiensis Endotoxin; Mycoplasmapulmonis ND; and Salmonella typhi OmpC porin. [See the references citedin J. B. Ulmer et al., ASM News, 62(9): 476-479 (1996).] Induction ofcytotoxic T lymphocytes (CTL), protective immunization, or both, hasbeen reported for each of these examples.

[0027] The potential advantages of DNA vaccines, in terms of efficacyand cost, over vaccines prepared by traditional approaches is consideredto be as significant as two other major advances that were developedover the past century. The first development pioneered by Louis Pasteurwas the use of attenuated and killed forms of microorganisms. The secondmajor development was the use of defined components of whole organismsand the use of purified recombinant proteins. The DNA vaccine approach,has been termed the “third vaccine revolution.” [B. Dixon,Bio/Technology, 13(5): 420 (1995).]

[0028] DNA vaccines offer several advantages over other types ofvaccines. [J. B. Ulmer et al., ASM News, 62(9): 476-479 (1996).] First,expression of antigen encoded on the vector introduced into host cellsleads to production of structurally relevant proteins, which areappropriately modified, and induction of cytotoxic T lymphocytes.Second, DNA vaccines induce CTL responses without resorting tocomplicated protein formulations or to attenuated live organisms.Attenuated live organisms, such as bacteria, or viruses, also have agreater inherent ability to mutate to more virulent forms. Third,expression of antigens after DNA vaccination can persist, for months insome cases, sufficient to promote the induction of memory immune cells.Still further, DNA vaccines can easily be injected intramuscularly orintradermally in simple aqueous solutions, or coated onto metalparticles which are blasted into cells with gene guns, which facilitatesadministration and their subsequent analysis. [Finan, E. F., et al.,Proc Natl Acad. Sci. U.S.A. 90: 11478-11482 (1993)].

[0029] The invention described hereinafter provides an autogeneicimmunological process for the production of antibodies to CETP and canprovide long-term lessening of transfer of cholesteryl esters from HDLparticles in mammals whose blood contains CETP by utilization of a DNAvaccine. This process permits the long-term elevation ofanti-atherogenic HDL cholesterol concentrations.

BRIEF SUMMARY OF THE INVENTION

[0030] The present invention contemplates an autogeneic immunologicalprocess for lessening the transfer of cholesteryl esters from HDLparticles and for increasing the HDL cholesterol concentration of amammal whose blood also contains CETP. A contemplated process is usefulin treating human pro-atherogenic dyslipoproteinemias characterized bylow HDL/LDL cholesterol ratios. Also contemplated here are isolated andpurified DNA that encode a useful immunogen and expression systems forthat DNA.

[0031] One contemplated process comprises the steps of:

[0032] (a) immunizing the mammal to be treated with an inoculumcontaining a DNA molecule that encodes a CETP immunogen that is animmunogenic polypeptide having a CETP amino acid residue sequence andwhich DNA molecule is dissolved or dispersed in a vehicle; and

[0033] (b) maintaining the immunized mammal for a time period sufficientfor the immunizing DNA to express the immunogenic polypeptide to inducethe production of antibodies that bind to CETP, and preferably alsolessen the transfer of cholesteryl esters from HDL. In one embodiment,the DNA encodes an immunogenic polypeptide that is an intact CETPmolecule such as recombinant human or rabbit CETP. In anotherembodiment, the encoded immunogenic polypeptide is a portion of a CETPmolecule that is covalently bonded to an exogenous antigenic carrier asa fusion protein.

[0034] In preferred embodiments, the exogenous antigenic carrier is thehepatitis B core protein (HBcAg) or diphtheria toxoid. HBcAg isparticularly preferred as an encoded exogenous antigenic carrier, thatforms a fusion protein with an immunogenic polypeptide having an aminoacid residue sequence of the carboxy-terminal 30 residues of CETP. Thatmore preferred fusion protein constitutes a polypeptide having the aminoacid residue sequence of the hepatitis B core antigen from which about 3to about 53 amino acid residues have been deleted and replaced by theimmunogenic polypeptide that more preferably still has a length aboutequal to the number of amino acid residues deleted from HBcAg. Theresulting fusion protein is most preferably expressed as particleshaving the size of HBcAg particles (about 27 nm).

[0035] The present invention has several benefits and advantages. Onesalient benefit is that a contemplated process can be utilized to lessenthe CE transfer from HDL to LDL or VLDL, thereby increasing theconcentration of anti-atherogenic HDL cholesterol.

[0036] An advantage of the invention is that a contemplated process canhave an effect that lasts for months as compared to the short-termeffects of the small molecule drugs now available.

[0037] Another benefit of a contemplated process is that it utilizes thehost mammal's own (autogeneic) immunological system to provide a desiredresult, thereby obviating problems associated with repeatedadministration of xenogeneic antibodies that themselves becomeimmunogenic in the host mammal.

[0038] Another advantage of some contemplated processes is that theiruse of well known and accepted exogenous antigenic carriers such asHBcAg, tetanus toxoid, and diphtheria toxoid can boost the host mammal'simmunity to those pathogens.

[0039] Still further benefits and advantages of the present inventionwill become apparent to a skilled worker from the disclosure thatfollows.

Definitions

[0040] The term “recombinant” is used to denote version of a DNA, RNA,or protein molecule altered with respect to the native molecule andresulting from the deletion, substitution, or insertion into the chain,by chemical, enzymatic, or biological means, of a sequence (a whole orpartial chain of DNA, RNA, or protein) not originally present in thatchain.

[0041] The term “recombinant DNA molecule” is used to mean a hybrid DNAsequence comprising at least two nucleotide sequences not normally foundtogether in nature.

[0042] The term “polypeptide” is used herein to denote a sequence ofabout 10 to about 500 peptide-bonded amino acid residues. A wholeprotein as well as a portion of a protein having the stated minimallength are polypeptides.

[0043] The term “fusion protein” is used to denote the expressionproduct of two or more different genes in which the amino acid residuesequences of both genes are expressed peptide-bonded together as asingle molecule. It is noted that a fusion protein need not have thefull length amino acid residue sequence of any protein, but ratherusually contains two or more truncated sequences. The term is thereforesomewhat of a misnomer, but is nonetheless well known and used asdefined here by those skilled in the art.

[0044] The term “fused”, when referring to expression of a fusionprotein, is used herein to mean peptide-bonded

[0045] The term “whole length CETP” is used to denote the full lengthCETP molecule (for example 476 amino acid residues long for human CETPor 496 residues long for rabbit CETP) as available in nature or producedas a recombinant protein.

[0046] The term “CETP immunogen” is used to denote molecule that is usedto induce the production of antibodies that immunoreact with (bind to)CETP.

[0047] The terms “immunogenic polypeptide having a CETP amino acidresidue sequence” or “immunogenic polypeptide” are used to denote theanti-CETP antibody-inducing portion of a “CETP immunogen”; i.e., thatportion of a CETP immunogen to which induced antibodies bind.

[0048] The term “exogenous antigenic carrier” or “carrier” is usedherein to denote a molecule foreign to the immunized mammal thatprovides a signal to antibody-producing B cells. Such carriers and theirfunctions are well known in the art. Such a carrier can be a polypeptidehaving a sequence of as few as about 10 amino acid residues to thelength of an intact protein, as well as being a synthetic polymer oroligomer.

[0049] The term “inoculum” in its various grammatical forms is usedherein to describe a composition containing an amount of CETP immunogen(e.g., DNA encoding a polypeptide conjugate, CETP protein or recombinantprotein) sufficient for a described purpose that is dissolved ordispersed in an aqueous, physiologically tolerable diluent.

[0050] The term “expression” is used to mean the combination ofintracellular processes, including transcription and translationundergone by a structural gene to produce a polypeptide.

[0051] The terms “operatively linked” or “operably inserted” are used tomean that two or more DNA sequences are covalently bonded together incorrect reading frame.

[0052] The term “promoter” is used to mean a recognition site on a DNAsequence or group of DNA sequences that provide an expression controlelement for a gene and to which RNA polymerase specifically binds andinitiates RNA synthesis (transcription) of that gene.

[0053] The term “structural gene” is used to mean a DNA sequence that isexpressed as a polypeptide; i.e., an amino acid residue sequence.

[0054] The term “vector” is used to mean a DNA molecule capable ofreplication in a cell and/or to which another DNA segment can beoperatively linked so as to bring about replication of the attachedsegment. A plasmid is an exemplary vector.

[0055] The term “expression vector” is used to mean a DNA sequence thatcauses a polypeptide to be expressed in that the DNA sequence containscontrol elements that regulate expression of structural genes whenoperatively linked to those genes within a vector.

DETAILED DESCRIPTION OF THE INVENTION

[0056] present invention relates to a process for producing antibodiesto CETP. Preferably, the produced antibodies lessen the transfer ofcholesteryl esters from HDL, and increase the ratio of HDL cholesterolto LDL cholesterol in the blood of a treated mammal that has CETP in itsblood. In humans, that increase in HDL to LDL ratio can lead to anamelioration of dyslipoproteinemias characterized by low HDL/LDLcholesterol ratios. That desired raising of the HDL/LDL cholesterolratio is accomplished immunologically by antibodies induced in the bloodof the treated mammal that recognize and bind to circulating CETP. Alsocontemplated in this invention are a DNA that encodes an immunogenutilized in the process, an inoculum that utilizes the DNA, and anisolated and purified DNA segment that encodes a contemplated immunogen.

[0057] I. The Process

[0058] A contemplated process is referred to herein as utilizing“autogeneic” antibodies to denote that the useful antibodies are thoseinduced in the host mammal itself. This autogeneic immunological processis therefore to be distinguished from a xenogeneic process in whichantibodies from an animal of one species are administered to an animalof another species as where mouse anti-CETP TP2 or 1C4 monoclonalantibodies have been administered to hamsters or rabbits. A contemplatedautogeneic immunological process is also to be distinguished from anallogeneic immunological process such as a passive immunization in whichantibodies from one animal are administered to another animal of thesame species as where humans receive gamma globulin injections fromother humans.

[0059] A contemplated process is thus closely analogous to an autoimmuneprocess in which a mammal's own immune system attacks an endogenous orself protein. CETP is an endogenous protein in rabbits, hamsters andprimates that are among the mammalian hosts contemplated here. However,inasmuch as the cause of most if not all autoimmune responses ispresently unknown and the desired immune response contemplated here ispurposefully induced, it is believed appropriate to use a different namefor the result obtained here.

[0060] One contemplated process produces antibodies to CETP in a mammal.That process comprises the steps of:

[0061] (a) immunizing the mammal with an inoculum containing a vehiclein which is dissolved or dispersed a recombinant DNA molecule comprisinga DNA sequence that contains (i) a sequence encoding a CETP immunogenlinked to (ii) a promoter sequence that controls the expression of theCETP immunogen DNA sequence in the mammal. The encoded CETP immunogen isan immunogenic polypeptide having a CETP amino acid residue sequence.The immunization provides an amount of the recombinant DNA moleculesufficient to be expressed and for the expressed immunogenic polypeptideto induce antibodies to CETP. The immunized mammal is (b) maintained fora time period sufficient for production of antibodies that bind to CETP.

[0062] Another aspect of the present invention contemplates a processfor lessening the transfer of cholesteryl esters from HDL particles andincreasing the concentration of HDL cholesterol in the blood of a mammalwhose blood contains cholesteryl ester transfer protein (CETP); i.e.,animals that have endogenous plasma CETP measured as a transferactivity. That process comprises the steps of: (a) immunizing thatmammal (the host) with an inoculum that contains a DNA-encoded CETPimmunogen and linked, controlling promoter sequence dissolved ordispersed in a vehicle. The DNA-encoded CETP immunogen is an immunogenicpolypeptide having a CETP amino acid residue sequence. The immunizedmammal is (b) maintained for a time period sufficient for the DNA toexpress the immunogenic polypeptide and for the expressed immunogenicpolypeptide to induce the production of antibodies that bind to CETP,and preferably lessen the transfer of cholesteryl esters (CE) from HDL.

[0063] A. The CETP-Encoded Immunogen

[0064] The immunizing DNA that encodes an immunogenic polypeptide havinga CETP amino acid residue sequence of the CETP immunogen can encode awhole CETP molecule such as the human (476 residues) or rabbit (496residues) proteins whose amino acid residue sequences are provided asSEQ ID NOs:28 and 26, respectively, and whose DNA sequences are providedin SEQ ID NOs:l and 27, respectively. The cDNA (SEQ ID NO:31) anddeduced amino acid residue sequence (SEQ ID NO:30) for cynomolgus monkeyCETP have also been reported by Pape et al., Atherosclerosis andThrombosis, 11:1759-1771 (1991). A DNA that encodes a polypeptide of SEQID NO:30 or a portion thereof as described below, can also be utilizedherein; i.e., the cDNA shown in SEQ ID NO:31 or a portion thereof,respectively.

[0065] Where the whole CETP molecule is encoded by the DNA as theimmunogenic polypeptide of the CETP immunogen, it is preferred to usethe DNA sequence that encodes a CETP sequence from an animal speciesother than that of the immunized mammal; i.e., the encoded CETP ispreferably xenogeneic as to the immunized mammal. When an encodedimmunogenic polypeptide is other than an intact CETP molecule, it ispreferred to use a DNA that encodes a polypeptide having a length ofabout 10 to about 30 amino acid residues, and more preferably, a lengthof about 20 to 30 residues. In this instance, the immunogenicpolypeptide is expressed covalently (peptide) bonded to an exogenousantigenic carrier to form the CETP immunogen. The encoded immunogenicpolypeptide and antigenic carrier sequences are, of course, linkedtogether in proper reading frame, as is an encoded immunogenicpolypeptide linked to the promoter when used without an antigeniccarrier.

[0066] Exogenous antigenic carrier polypeptide molecules are also wellknown in the art, as are the amino acid residue and nucleotide sequencesof those molecules. Exemplary polypeptide carriers include but are notlimited to tetanus toxoid, diphtheria toxoid, thyroglobulin and thehepatitis B core protein (HBcAg).

[0067] Thus, the cDNA encoding an exogenous antigenic carrier and thatencoding an immunogenic CETP polypeptide are operatively linked to forma single isolated and purified DNA molecule that encodes both thecarrier and immunogenic polypeptide. That DNA molecule can then beoperatively linked in an appropriate expression vector along with apromoter that controls expression of those two polypeptides as a singlefusion protein whose two polypeptide portions are covalently bonded by apeptide bond. Preferably, the carrier is expressed at the amino-terminusof the fusion protein, although a carrier can also be expressed fused atthe carboxy-terminus of the immunogenic polypeptide. Exemplary proteinsand procedures for their synthesis are discussed hereinafter.

[0068] Preferably, where the whole CETP molecule is used as theimmunogenic polypeptide, the carrier polypeptide has an amino acidresidue sequence that is less than that of a whole protein. That lengthis preferably about 15 to about 70 amino acid residues.

[0069] The hepatitis B nucleocapsid or core protein antigen alsoreferred to as HBcAg is a particularly preferred exogenous antigeniccarrier, as will be discussed in greater detail hereinafter. The HBcAgmolecule is often used illustratively herein as a carrier.

[0070] U.S. Pat. No. 4,818,527, whose disclosures are incorporated byreference, teaches that the region extending from about position 70through about position 140 from the amino-terminus of HBcAg, whosecomplete amino acid and cDNA sequences are shown as SEQ ID NOs:38 and39, respectively, is particularly useful as a T cell independentstimulant as are sequences of about 15 to about 25 residues from thatregion. The amino acid residue sequences of four of those shorterpolypeptides are provided as SEQ ID NOs:40, 41, 42 and 43. The cDNAsequences that encode each of those four polypeptides can be readilyobtained from SEQ ID NO:39, and the 3′ end of such a cDNA can beoperatively linked to the 5′ end of cDNA that encodes an immunogenicpolypeptide, or vice versa, for expression as a fusion protein CETPimmunogen.

[0071] Thus, in one embodiment, a preferred recombinant DNA molecule(defined previously and discussed hereinafter) encodes a CETP immunogenthat is a fusion protein whose amino-terminal portion is a polypeptidehaving a length of about 15 to about 70 amino acid residues and havingthe sequence of HBcAg from about position 70 to about position 140 fromthe HBcAg amino-terminus. The carboxy-terminal portion of that fusionprotein has the amino acid residue sequence of a CETP molecule, and thetwo portions are covalently bonded by a peptide bond. In thisembodiment, the CETP molecule can be from the same species as theimmunized mammal.

[0072] In another preferred embodiment, the immunizing DNA encodes aCETP immunogen that is comprised of an exogenous antigenic carrier towhich one or more immunogenic polypeptides having a length of about 10to about 30 amino acid residues such as those of SEQ ID NOs:2-7 or 50having a sequence of rabbit CETP, the similar polypeptides of SEQ IDNOs:8-13 or 29 having a sequence of human CETP or the similarpolypeptides of SEQ ID NOs:32-37 having a sequence of monkey CETP iscovalently bonded. Here, the carrier is preferably an intact proteinsuch as a before-noted tetanus toxoid, diphtheria toxoid, thyroglobulinor HBcAg molecule.

[0073] As noted before, a DNA sequence for the CETP molecule or adesired portion thereof can be obtained as described by M. E. Pape etal., Arteriosclerosis and Thrombosis, 11:1759-1771 (1991); N. W. Jeonget al., Mol. Cells, 4(4):529-533 (1994); and D. T. Connolly et al.,Biochem. J., 320:39-47 (1996). Oligonucleotides can also be preparedusing standard synthetic technology where shorter DNA sequences aredesired. Those oligonucleotides can also be linked enzymatically, aswith T4 DNA ligase, to form longer molecules.

[0074] DNA sequences for exogenous antigenic carrier molecules have alsobeen reported as have methods for expressing those molecules. Forexample, a DNA sequence that encodes the preferred HBcAg exogenousantigenic carrier is disclosed in U.S. Pat. No. 4,710,463, whosedisclosures are incorporated herein by reference, and E. coli-containingplasmids whose DNA encode hepatitis B virus proteins were deposited inthe Culture Collection of the National Collection of IndustrialBacteria, Aberdeen Scotland as pBR322-HBV G-L. In addition, DNA encodingHBcAg is disclosed in U.S. Pat. No. 4,942,125 as present in vectorsdeposited at the American Type Culture Collection (ATCC), 12301 ParklawnDrive, Rockville, Md. 20852-1776 as ATCC No. 39629, No. 39631 and No.40102.

[0075] The use of HBcAg as an exogenous antigenic carrier in a fusionprotein is illustrated in Moriarty et al., Vaccines 90, Brown et al.eds., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.,225-229 (1990). The authors there reported operatively linking the 3′end of DNA encoding a 17-mer amino acid residue sequence of the HIV gagprotein to the 51 end of DNA encoding HBcAg, and reported thatappropriately transfected E. coli expressed a fusion protein having theHIV gag sequence peptide-bonded to the amino-terminus of HBcAg. Thatexpressed fusion protein was present in particulate form and was shownto be an excellent immunogen in mice.

[0076] Schödel et al., Vaccines 90, Brown et al. eds., Cold SpringHarbor Laboratory Press, 193-198 Cold Spring Harbor, N.Y. (1990)reported the preparation and successful use of a fusion proteinimmunogen that contained a polypeptide immunogen having an amino acidresidue sequence of hepatitis B Pre-S2 (residues 133-140) that wasexpressed peptide-bonded to the carboxy-terminus of HBcAg so that the 3′end of the exogenous carrier (HBcAg) DNA was linked to the 5′ end of theDNA that encoded the Pre-S2 polypeptide immunogen. That expressed fusionprotein immunogen was also obtained in particulate form.

[0077] Similar techniques can be utilized here using a CETP-encodingrecombinant DNA molecule that contains a DNA molecule of SEQ IDNOs:14-19, 20-25 or a corresponding DNA sequence of SEQ ID NO:31 thatencodes a CETP immunogenic polypeptide in place of the DNAs used by theMoriarty et al. or the Schödel et al. groups.

[0078] In addition, using similar techniques and others well known toworkers of ordinary skill in the recombinant DNA art, a DNA moleculethat encodes a fusion protein can be prepared that expresses apolypeptide having an HBcAg amino acid residue sequence such as one ofthose of SEQ ID NOs:40-43 peptide-bonded to the amino-terminus of anintact CETP molecule.

[0079] A particularly preferred DNA-encoded CETP immunogen is a fusionprotein comprised of an immunogenic polypeptide having a length of 10 toabout 30 amino acid residues that is peptide-bonded to both anamino-terminal flanking amino acid residue sequence and acarboxy-terminal flanking sequence, and is sometimes referred tohereinafter HBcAg/CETP/HBcAg. Those flanking sequences are preferablyportions from the amino-terminal and carboxy-terminal regions of theHBcAg molecule, as was discussed previously. Thus, in this fusionprotein, the exogenous antigenic carrier molecule is encoded to bepeptide-bonded to both the amino-terminus and carboxy-terminus of theimmunogenic polypeptide.

[0080] A preferred encoded polypeptide immunogen (immunogenicpolypeptide) has an amino acid residue sequence of SEQ ID NOs:2-7, 8-13,29, 32-37 or 50. Most preferably, the encoded polypeptide immunogen hasan amino acid residue sequence that is bound by (immunoreacts with) themonoclonal antibodies designated TP1, TP2 and TP3 reported by B. Hessleret al., J. Biol. Chem., 263(11):5020-5023 (1988), or that denominated1C4 by J. Gaynor et al., Atherosclerosis, 110(1):101-109 (1994).Monoclonal antibody TP2 binds to an epitope located between aboutpositions 465 and 475 of human CETP. Tall, J. Lipid Res., 34:1255-1274(1993), and the citations therein.

[0081] A particularly preferred recombinant DNA molecule contains acontrolling promoter sequence linked to an encoded polypeptide immunogensequence whose encoded amino acid residue sequence includes positions465 through 475 of human CETP or an analogous position of CETP fromanother source. An encoded polypeptide is exemplified by those of SEQ IDNOs:4, 10, 29 and 34, of which the encoded polypeptides of SEQ ID NOs:10and 29 are most preferred. The polypeptide of SEQ ID NO:10 is encoded bythe DNA of SEQ ID NO:22.

[0082] Protein molecules have not only a linear amino acid residue orprimary sequence, but also can possess a secondary sequence in which thepolypeptide back bone is coiled in an α-helix or folded into a β-sheet,as well as a tertiary sequence in which sequentially distant portions ofthe molecule are folded to be adjacent to each other. Many linearantigenic/immunogenic polypeptide sequences have been reported in theliterature, and such sequences can be readily mimicked by polypeptideshaving a length of 10 to about 30 amino acid residues. Such relativelyshort polypeptides typically do not mimic a secondary structure such asan α-helix in aqueous media.

[0083] The region of CETP that immunoreacts with monoclonal antibody TP2is predicted to have an amphipathic helical secondary structure, withthe hydrophilic surface bound by the antibody. See Wang et al., J. Biol.Chem., 267(25) :17487-17490 (1992) and A. R. Tall, J. Lipid Res.34:1255-1274 (1993). A contemplated DNA encodes a CETP immunogen fusionprotein having an immunogenic polypeptide flanked at its amino- andcarboxy-termini by peptide-bonded regions of HBcAg; i.e.,HBcAg/CETP/HBcAg, that is more constrained in its molecular motions thanis an immunogenic polypeptide that is bonded at only one terminus. As aconsequence, by flanking a before-mentioned particularly preferredimmunogenic polypeptide with regions of HBcAg to form a HBcAg/CETP/HBcAgfusion protein, it is believed that the expressed immunogenicpolypeptide becomes constrained to have a helical structure much likethat present in the native CETP molecule and thereby induce autogeneicantibodies having an antigenic specificity similar to those exhibited bymouse monoclonal antibodies TP1, TP2, TP3 and 1C4 discussed previously.

[0084] It is further believed that formation of HBcAg-like particles ofan expressed fusion protein HBcAg/CETP/HBcAg immunogen places furtherconformational constraints upon the immunogenic polypeptide by which theimmunogenic polypeptide becomes the primary immunogen with loss of muchof the HBcAg immunogenicity, while the T cell-independent antigeniccarrier function of HBcAg is retained. See Schödel et al., J. Virol.,66(1):106-114 (1992) for a similar result using a different immunogen.

[0085] Although use of the full length HBcAg exogenous antigenic carriermolecule or substantially full length molecule has thus far beendiscussed, it is noted that about 10 amino-terminal amino acid residues(about 30 base pairs) or about 40 carboxy-terminal amino acid residues(about 120 base pairs) can be deleted from the expressedHBcAg/CETP/HBcAg sequence (encoding DNA) without abrogating function asan exogenous antigenic carrier or assembly into particles. See, forexample, Birnbaum et al., J. Virol., 64(7):3319-3330 (1990).

[0086] Exemplary preparations of immunogenic fusion proteins havingHEcAg as a carrier with various heterologous polypeptide insertions frompathogens as immunogen, and also usage of full length andcarboxy-terminal deletions in the HBcAg amino acid residue sequence canbe found in the following publications. Schödel et al., J. Exp. Med.,180:1037-1046 (1994); Schödel et al., J. Virol., 66(1):106-114 (1992);Schödel et al., Vaccines 91, Brown et al. eds., Cold Spring HarborLaboratory Press, Cold Spring Harbor, N.Y., 319-325 (1991); Clarke etal., Vaccines 91, Brown et al. eds, Cold Spring Harbor Laboratory Press,Cold Spring Harbor, N.Y., 313-318 (1991); and Schödel et al., Vaccines90, Brown et al. eds., Cold Spring Harbor Laboratory Press, Cold SpringHarbor, N.Y., 193-198 (1990).

[0087] It is also noted that the human hepatitis virus (HBV), whose coreantigen is discussed herein, has two subtypes that are denominated adwand ayw. The core antigens of those two viral subtypes have slightlydifferent DNA and amino acid residue sequences. Although subtypespecificity has been noted as to the immunogenicity of the S and PreSregions of HBV, [see, for example, Milich et al. Vaccines 86, Brown etal. eds., Cold Spring Harbor Press, Cold Spring Harbor, N.Y., 377-382(1986)] either subtype can be used as an exogenous antigenic carrierherein, with subtype ayw being used illustratively herein.

[0088] It should also be noted that although a contemplated process hasto a great extent been discussed in terms of the polypeptide immunogenthat is ultimately expressed and induces autogeneic antibody productionto CETP, that immunogenic polypeptide may not be readily discernable inan immunized host mammal. The DNA that encodes such an immunogenicpolypeptide may also not be easily identifiable in the mammalian hostwhere no reporter gene such as β-galactosidase is included as a part ofthe immunizing recombinant DNA molecule, or where an immunizingrecombinant DNA molecule, which exists and functions extrachromosomally,is eliminated from an immunized cell at a time subsequent to expressionof a CETP immunogen. Antibodies that bind to CETP are, however, presentin the host after an appropriate amount of time, and the presence ofthose antibodies provides evidence that a contemplated process has beencarried out in that such CETP-binding antibodies are not known to arisenaturally.

[0089] B. DNA Molecules and Expression Systems

[0090] A contemplated DNA molecule (isolated purified DNA segment) thatencodes a CETP immunogen can be referred to as a number of base pairs ata particular location in a plasmid, as a restriction fragment bounded bytwo restriction endonuclease sites, and as a restriction fragmentbounded by two restriction endonuclease sites and containing a number ofbase pairs. A contemplated DNA can also be defined to have a sequence ofa denominated SEQ ID NO, as well as alleles or variants of such genes(described hereinafter) that encode a recited amino acid residuesequence.

[0091] A contemplated isolated and purified DNA segment is linear, andas such has a 5′ end and a 3′ end. A contemplated DNA segment cancomprise two or more individual DNA segments whose 3′ ends areoperatively linked to the 5′ end of another DNA segment where twosegments are joined, or whose 3′ end is operatively linked to the 5′ endof another DNA segment whose own 3′ end is operatively linked to the 5′end of yet another DNA segment, where three individual segments arejoined to form a single isolated and purified DNA segment.

[0092] In living organisms, the amino acid residue sequence of a proteinor polypeptide is directly related via the genetic code to thedeoxyribonucleic acid (DNA) sequence of the structural gene that codesfor the protein. A structural gene can be defined in terms of the aminoacid residue sequence; i.e., protein or polypeptide, for which it codes.

[0093] In addition, through the well-known redundancy of the geneticcode, additional DNA sequences can be prepared that encode the sameamino acid residue sequences, but are different from a recited genesequence having a particular SEQ ID NO. For example, in vitromutagenesis as is illustrated hereinafter can be used to change a DNAsequence so that the same residue of an expressed polypeptide isexpressed using one or more different codons. In addition, that sametechnique can be used to change one amino acid residue to another whereit is desired to insert or delete specific restriction endonucleasesites. This technique is also illustrated hereinafter.

[0094] A DNA sequence that encodes a CETP immunogen of a recited SEQ IDNO but has a DNA sequence different from that of a recited SEQ ID NO isreferred to herein as a variant DNA sequence. Such a variant DNAmolecule can be readily prepared by in vitro mutagenesis, as is wellknown.

[0095] A DNA segment that encodes a described CETP immunogen can besynthesized by chemical techniques, for example, the phosphotriestermethod of Matteucci et al., J. Am. Chem. Soc., 103:3185 (1981). Ofcourse, by chemically synthesizing the coding sequence, any desiredmodifications can be made simply by substituting the appropriate basesfor those encoding the native amino acid residue sequence.

[0096] However, DNA segments including the specific sequences discussedpreviously are particularly preferred. Furthermore, a DNA segment thatencodes a polypeptide can be obtained from a recombinant DNA molecule(plasmid or other vectors) containing that segment.

[0097] A DNA segment that includes a DNA sequence encoding a CETPimmunogen can be prepared by excising and operatively linkingappropriate restriction fragments from appropriate plasmids or other DNAusing well known methods. The DNA molecules useful here that areproduced in this manner typically have cohesive termini; i.e.,“overhanging” single-stranded portions that extend beyond thedouble-stranded portion of the molecule. The presence of cohesivetermini on the DNA molecules of the present invention is preferred,although molecules having blunt termini are also contemplated.

[0098] A recombinant DNA molecule useful herein can be produced byoperatively linking a vector to an isolated DNA segment that encodes aCETP immunogen to form a plasmid such as those discussed herein.Particularly preferred recombinant DNA molecules are discussed in detailin the examples, hereafter. Vectors capable of directing the expressionof the gene in the immunized mammal are referred to herein as“expression vectors”.

[0099] The expression vectors described above contain expression controlelements including a promoter. The genes that encode an immunogenicpolypeptide or other useful sequence are operatively linked to theexpression vector to permit the promoter sequence to direct RNApolymerase binding and expression of the desired polypeptide codinggene.

[0100] The choice of which expression vector to which apolypeptide-coding gene is operatively linked depends directly on thefunctional properties desired, e.g. the location and timing of proteinexpression, and the host cell to be transformed. These are well knownlimitations inherent in the art of constructing recombinant DNAmolecules. However, a vector useful in practicing the present inventionis capable of directing the replication and also the expression of theimmunogenic polypeptide-coding gene included in the DNA segment to whichit is operatively linked.

[0101] A cloning vector is also useful herein for making or increasingthe amount of a desired DNA, and can also be used to express animmunogenic polypeptide to assay the synthesized DNA. A cloning vectorincludes a prokaryotic replicon; i.e., a DNA sequence having the abilityto direct autonomous replication and maintenance of the recombinant DNAmolecule extrachromosomally in a prokaryotic host cell transformedtherewith. Such replicons are well known in the art as are cloningvectors, some of which are discussed below.

[0102] Those vectors that include a prokaryotic replicon can alsoinclude a prokaryotic promoter region capable of directing theexpression of the CETP immunogen gene in a host cell, such as E. coli,transformed therewith. Promoter sequences compatible with bacterialhosts are typically provided in plasmid vectors containing one or moreconvenient restriction sites for insertion of a DNA segment of thepresent invention. Such cloning and expression vector plasmids are wellknown in the art. Typical of such cloning and expression vector plasmidsare pUC18, pUC19, pBR322, pProEx1, and pFastBac1 available from LifeTechnologies, Rockville, Md., and pPL and pKK223-3 available fromPharmacia, Piscataway, N.J. These vectors are utilized in the synthesisof the DNA segments useful herein.

[0103] In preferred embodiments, the cloning vector used to express animmunogenic polypeptide-coding gene includes a selection marker that iseffective in a host cell, preferably a drug resistance selection marker.One preferred drug resistance marker is the gene whose expressionresults in kanamycin resistance, whereas ampicillin resistance isanother such marker. Again, such selective markers are well known.

[0104] A variety of methods has been developed to operatively link DNAto vectors via complementary cohesive termini or blunt ends. Forinstance, complementary homopolymer tracts can be added to the DNAsegment to be inserted and to the vector DNA. The vector and DNA segmentare then joined by hydrogen bonding between the complementaryhomopolymeric tails to form recombinant DNA molecules.

[0105] Alternatively, synthetic linkers or adapters containing one ormore restriction endonuclease sites can be used to join the DNA segmentto the integrating expression vector. The synthetic linkers or adaptersare attached to blunt-ended DNA segments by incubating the blunt-endedDNA segments with a large excess of synthetic linker or adaptermolecules in the presence of an enzyme that is able to catalyze theligation of blunt-ended DNA molecules such as bacteriophage T4 DNAligase.

[0106] Thus, the products of the reaction are DNA segments carryingsynthetic linker sequences at their ends. These DNA segments are thencleaved with the appropriate restriction endonuclease and ligated intoan expression vector that has been cleaved with an enzyme that producestermini compatible with those of the synthetic linker. Synthetic linkerscontaining a variety of restriction endonuclease sites are commerciallyavailable from a number of sources including New England BioLabs,Beverly, Mass. A synthetic adapter molecule typically has sticky end andone blunt end and is not cleaved after ligation.

[0107] Although preferred, it is not always feasible to design a DNAmolecule whose expressed polypeptide has the exact terminal residues ofa polypeptide enumerated in a SEQ ID NO. This is because of thelimitations inherent in the use of restriction enzymes, syntheticlinkers and adapter molecules used for cutting and joining DNA segments.

[0108] As a consequence, an expressed polypeptide can contain a few(e.g., one or two) more, less or different amino acid residues at one orboth termini of an enumerated sequence. Such slight changes are welltolerated by a contemplated CETP immunogen, particularly when thesubstitution is conservative and residues such as Cys and Pro areavoided.

[0109] A variety of plasmids can be used as DNA vaccine vectors forexpressing a contemplated CETP immunogen in a mammalian host. Suchvectors optimally include the following components: a strong eukaryoticpromoter, a cloning site for insertion of a gene of interest, apolyadenylation termination [poly(A)] sequence, a prokaryotic origin ofreplication, and a prokaryotic selectable marker. One such vector, pV1J,contains the cytomegalovirus immediate-early promoter with intron A, abovine growth hormone polyadenylation termination sequence, and anampicillin resistance gene. [J. B. Ulmer et al., ASM News, 62(9):476-479 (1996).] Another useful vector denominated pcDNAI/Amp that isavailable from Invitrogen, Corp. of San Diego, Calif., as well asplasmids pCMV-SPORT-β-gal and pGreen Lantern-1 available from LifeTechnologies, Rockville, Md. are discussed in detail hereinafter. Othereukaryotic promoters, poly(A) sites, and selectable markers can besubstituted without departing from the utility of the vector, as long asthe structural gene inserted downstream from the promoter is expressedin mammalian cells. A variety of general mammalian expression vectors,many of which are commercially available, are suitable for use herein asDNA vaccine vectors.

[0110] Plasmid DNA can be prepared by a variety of methods. High qualityplasmid DNA can be prepared using CsCl gradients to separate covalentlyclosed circular plasmid DNA from linearized plasmid and chromosomal DNA.Other components from lysed bacterial cells such as proteins, RNA,membranes, and cell wall material are generally well-separated from theplasmid DNA by density gradient centrifugation. Standard protocols forCsCl purification are well known in the art. [See, J. Sambrook et al.,Molecular Cloning, 2nd ed., Cold Spring Harbor Press, Cold Spring HarborN.Y., 8-23 (1989).]

[0111] Another method suitable for preparation of high quality plasmidDNA is anion exchange chromatography. Direct comparisons of DNA preparedby anion exchange chromatography and DNA banded twice on CsCl gradientsreportedly showed no difference in the efficiency of direct genetransfer and genetic immunization for reporter plasmids. [H. L. Davis etal., Biotechniques, 21: 99-99 (1996).] DNA banded once on CsCl gradientswas reported to contain less double-stranded closed circular DNA andmore RNA contamination than DNA banded twice on these gradients.Endotoxin levels were said to greater, however, for DNA purified byanion exchange chromatography than by CsCl purification. [H. L. Davis etal., Biotechniques, 21: 99-99 (1996).]

[0112] A preparative method of purification of supercoiled plasmid DNAfor therapeutic applications can also be used [A. P. Green et al.,Biopharm, 10(5): 52-62, (1997)]. This method uses ion-pairing reversedphase column chromatography to prepare plasmid DNA free fromcontaminating DNA, RNA, protein, and endotoxin.

[0113] General methods and procedures for characterizing bulk plasmidDNA vectors suitable for use in human gene therapy have been described[M. Marquet et al., BioPharm, 10(5): 42-50 (1997)]. Methods tocharacterize a master cell bank, by testing plasmid identity, plasmidyield, plasmid stability, and methods to verify the genotype of the hoststrain and to measure host strain viability and microbial contaminationwere described. Methods to characterize the bulk product in terms ofsterility and for the presence of pyrogens were also described.

[0114] Endotoxin is the lipopolysaccharide component of the cell wall ofgram-negative bacteria that is released when cells are lysed forrecovery of plasmid DNA. Endotoxin can have cytotoxic effects onmammalian cells in vitro or in vivo. [I. P. Wicks et al., Hum. GeneTher., 6: 317-323 (1995)]. Endotoxin levels can be measured by achromogenic limulus amoebocyte clotting assay [SCL-100 kit;BioWhittacker, Walkerville, Md.; T. Mossman, J. Immunol. Meth., 65:55-63 (1983)]. Methods for purification of endotoxin-free plasmid DNAprepared by anion exchange chromatography are now available. [J. Schorret al., Gene Ther., 1: S7 (1994)]. This method clears the bacteriallysate with a special endotoxin removal buffer before the DNA ispurified from lysate on an anion exchange column.

[0115] A plasmid or other vector DNA that encodes a contemplated CETPimmunogen and contains a promotor sequence as discussed before thatcontrols expression of that immunogen-encoding DNA sequence in properreading frame comprises a recombinant DNA molecule utilized inimmunizing a host mammal. That recombinant DNA molecule dissolved ordispersed in a vehicle comprises an inoculum that is used to immunizethe mammal and is discussed further hereinbelow.

[0116] C. Inocula

[0117] A DNA vaccine vector encoding a CETP immunogen in proper readingframe (a recombinant DNA molecule) and containing a promoter sequencethat controls expression of the immunogenic polypeptide is dissolved ordispersed in a pharmaceutically-acceptable vehicle composition that ispreferably aqueous to form an inoculum that when used to immunize amammal induces the production of antibodies that immunoreact with (bindto) CETP. When that recombinant DNA molecule is administered in aneffective amount to a mammal whose blood contains CETP those antibodiespreferably also lessen the transfer of cholesteryl esters from HDLparticles.

[0118] An effective recombinant DNA molecule dosage is typically about0.05 μg/kg to about 50 mg/kg, usually about 0.005 mg/kg to about 5mg/kg. Methods of determining the effective systemic dose, which dosecan vary depending on the activity of the polypeptide encoded by the DNAcan be determined in a manner apparent to those of skill in the art.U.S. Pat. No. 5,580,859, whose disclosure is herein incorporated byreference, discloses several methods of determining an effective dose ofa DNA vaccine.

[0119] The term “unit dose” as it pertains to an inoculum of the presentinvention refers to physically discrete units suitable as unitarydosages for animals, each unit containing a predetermined quantity ofactive material calculated to individually or collectively produce thedesired immunogenic effect in association with the required diluent;i.e., carrier, or vehicle.

[0120] Inocula are typically prepared from a recombinant DNA moleculeencoding a CETP immunogen by dispersing the DNA in a liquidphysiologically tolerable (acceptable) diluent vehicle such as water, orphosphate-buffered saline (PBS), or the like to form an aqueouscomposition. The amount of DNA-encoding CETP immunogen utilized in eachimmunization can vary widely, and is referred to as an effective amount.Such an effective amount is sufficient to induce antibodies to CETP thatbind to CETP, and preferably lessen the transfer of cholesteryl estersfrom HDL particles, and also increase the HDL/LDL ratio in the immunizedmammal's blood. Exemplary effective amounts of a DNA-encoded CETPimmunogen are about 0.005 mg/kg to about 5 mg/kg, depending inter alia,upon the sequence of the encoded CETP immunogen, the mammal immunized,and the presence of salts, stabilizers, and cell penetration agents inthe inoculum. An exemplary unit does can constitute about 10 μl to about1 ml per site of administration (immunization), with a recombinant DNAmolecule concentration of about 0.05 μg/ml to about 20 mg/ml, andpreferably about 0.1 μg/ml to about 100 μg/ml. Thus, a single unit doseor a plurality of unit doses can be used to provide an effective amountof expressed CETP immunogen. Those skilled in the art know thatappropriate concentrations or amounts can be readily determined.

[0121] An inoculum is typically formulated for parenteraladministration. Exemplary immunizations are carried out sub-cutaneously(s.c.) intramuscularly (i.m.) or intra-dermally (i.d.). U.S. Pat. No.5,580,859, discloses several formulations suitable for use with a DNAvaccine. These include inocula composed of recombinant DNA moleculesdissolved or dispersed in an aqueous vehicle containing salts, diluents,stabilizers, and cell penetration agents.

[0122] Typical formulations include recombinant DNA molecules suspendedin phosphate buffered saline or isotonic sucrose. DNA can also becomplexed with cell penetration agents such as liposomes, whichfacilitate uptake of the DNA into cells. One example of a liposome whichhas been used in DNA vaccine formulations is Lipofectin™, availablecommercially from Life Technologies, Rockville, Md. Uptake andexpression are often also significantly enhanced if DNA is administeredin conjunction with the facilitating agent bupivacaine-HCl (ananesthetic) [Coney, L., et al., Vaccine, 12: 1545-1550 (1994)]. Thoseskilled in the art will recognize that other similar cell penetrationand facilitating agents can be used for these purposes and can thatappropriate concentrations or amounts of these agents can be readilydetermined.

[0123] Once immunized, the mammal is maintained for a period of timesufficient for the encoded CETP immunogen to be expressed and then forthe expressed CETP immunogen to induce the production of antibodies thatbind to CETP and lessen the transfer of cholesteryl esters from HDLparticles. This maintenance time typically lasts for a period of aboutthree to about eight weeks, and can include a booster, second immunizingadministration of the inoculum.

[0124] The production of antibodies that bind to CETP is readilyascertained by obtaining a plasma or serum sample from the immunizedmammal and assaying the antibodies therein for their ability to bind toCETP as an antigen in an ELISA assay as described hereinafter, or byanother immunoassay such as a Western blot as is well known in the art.

[0125] The lessening of transfer of cholesteryl esters from HDL can beassayed by one or more of several techniques. In one assay, the rate oftransfer is measured by use of a [³H]-cholesteryl ester ([³H]CE) fromHDL to LDL following the differential precipitation assay reported byGlenn et al., Methods in Enzymology, 263:339-350 (1996). Briefly, in avolume of 200 μl, CETP, [³H]CE-labeled HDL, LDL, and TES assay buffer(50 mM Tris, pH 7.4; 150 mM NaCl; 2 mM EDTA; 1% bovine serum albumin)are incubated for 2 hours at 37° C. in 96-well filter plates. LDL isthen differentially precipitated by the addition of 50 μl of 1% (w/v)dextran sulfate/0.5 M MgCl₂. After filtration, the radioactivity presentin the precipitated LDL is measured by liquid scintillation counting.Correction for non-specific transfer or precipitation is made byincluding samples that did not contain CETP. The rate of [³H]CE transferis determined in the linear range of the assay with respect to time andCETP concentration. For studies in which antibodies are included in theassay, the order of addition into sample wells is: buffer,[³H]CE-labeled HDL, LDL, antibodies, CETP.

[0126] CETP activity can also be measured using two methods that do notinvolve differential precipitation. In the first assay, the incubationconditions are identical to those described above, but separation of LDLacceptor particles from [³H]CE-labeled HDL donor particles isaccomplished by size exclusion chromatography on tandem columns ofSuperose™ 6 (Sigma Chemical Co.), followed by liquid scintillationcounting of fractions to determine the amount of [³H]CE associated withLDL and HDL. The amount of transfer measured by this method is typicallyin excellent agreement with the precipitation assay.

[0127] Another assay for CETP activity measures the rate ofCETP-mediated transfer of the fluorescent analog NBD-cholesteryllinoleate (NBD-CE) from an egg phosphatidyl choline emulsion to VLDL.This assay takes advantage of the fact that NBD-CE is self-quenched whenin the emulsion, and becomes fluorescent when transferred to VLDL. Theassay is carried out according to the manufacturer's instructions(Diagnescent Technologies Inc., Yonkers, N.Y.). Fluorescencemeasurements can be taken using a standard machine such as an SLM 8000Cspectrophotofluorometer (Milton Roy Co., Rochester, N.Y.) using 465 nmand 535 nm for excitation and emission wavelengths, respectively.

[0128] It is particularly contemplated once the desired antibodies areinduced in the mammal that the immunization step be repeated atintervals of about 3 to about 6 months until the HDL cholesterol valuein the blood of the mammal is increased by about 10 percent or morerelative to the HDL cholesterol value for the mammal prior to the firstimmunization step. Preferably, the HDL cholesterol value is increased byabout 25 percent. The mammal is thereafter preferably maintained at thatincreased HDL cholesterol level by periodic booster immunizationsadministered at intervals of about 6 to about 18 months. The increase inHDL cholesterol can be measured by any reliable assay, many of which arewell known in the art, and one of which is described hereinafter.

[0129] It is noted that the before-described anti-CETP antibodies soinduced can be isolated from the blood of the host mammal using wellknown techniques, and then reconstituted into a second inoculum forpassive immunization as is also well known. Similar techniques are usedfor gamma-globulin immunizations of humans. For example, antiserum fromone or a number of immunized hosts can be precipitated in aqueousammonium sulfate (typically at 40-50 percent of saturation), and theprecipitated antibodies purified chromatographically as by use ofaffinity chromatography in which CETP or an immunogenic polypeptideportion thereof is utilized as the antigen immobilized on thechromatographic column.

BEST MODE FOR CARRYING OUT THE INVENTION

[0130] Comparative Examples 1 and 2, below, illustrate results obtainedusing a CETP polypeptide immunogen prepared exogenously to the immunizedmammal.

Comparative Example 1 Immunization of Rabbits With Rabbit CETP-Peptides

[0131] There is a 88 percent homology between rabbit and human CETP atthe amino acid residue level. Rabbits express high levels of CETP intheir blood and were chosen as a model for illustrating production ofautogeneic anti-CETP antibodies.

[0132] The six rabbit CETP polypeptides of SEQ ID NO:2-7 were selectedfor this study and were prepared by standard solid phase synthesisprocedures discussed below. To enhance the anti-polypeptide-specificantibody responses, two separate immunization strategies were used withthe above six rabbit CETP-polypeptides.

[0133] A. Immunization Strategy 1 (MAP conjugates)

[0134] Rabbit polypeptides were synthesized as multiple antigenicpeptide (MAP) constructs [D. N. Posnett et al., J. Biol Chem.,263:1719-1725 (1988)]. Those polypeptides were separately covalentlybonded to “oligolysine core” molecules that were themselves covalentlyattached to resin particles [S. Butz et al., Pep. Res. 1:20-223 (1994)].

[0135] The substitution of the starting resin particles was 0.37 μmsites/mg resin that provided approximately 500 μg of immunogenicpolypeptide per 1.1 mg resin. For the preparation of the CETP immunogenfor immunization, 3.0 mg of dry resin were weighed out and hydrated in1.3 ml sterile phosphate-buffered saline (PBS; pH 7.4) to which 1.3 mlFreund's complete adjuvant (CFA; Sigma Chemical Co., St. Louis, Mo.,F-5881) were added as adjuvant. The CETP immunogen and adjuvant wereemulsified by a female-female luer lock syringe adapter connected to two3 ml syringes. Each final emulsion was divided into 1.0 ml aliquots forinjection (1 ml/rabbit), with one immunogen used per rabbit. Pre-immunerabbit serum was collected before immunization and stored at −70° C.until immunoassay. On day 1, New Zealand white rabbits were separatelyimmunized with respective immunogens by sub-cutaneous (s.c.) route onthe back of the rabbit using 10 injection sites.

[0136] Three weeks later (on Day 22), the rabbits were boosted usingsimilar procedures, but this time CETP immunogens were emulsified inFreund's incomplete adjuvant (IFA; Sigma). The resin-bonded CETPimmunogen was weighed out as before and hydrated with sterile PBS theday before the booster immunization. The resulting CETP immunogensuspension was sonicated with a microtip at maximum setting for 5minutes and left overnight (about 18 hours) at 4° C. Before mixing thehydrated CETP immunogen suspension with IFA, the suspension was warmedto room temperature just before the booster immunization, added to 1.5ml IFA, and emulsified as described above to form an inoculum in whichthe CETP immunogen was dispersed. Rabbits were immunized each with 1 mlof emulsion in at least 10 injection sites S.C.

[0137] The first post-immune serum was collected 2 weeks after thesecond immunization from each animal. All the anti-sera samples werestored in −70° C. until ELISA was done.

[0138] Using this MAP strategy, polypeptides of SEQ ID NOs:2 and 7 weremoderately immunogenic in rabbits and resulted in maximum autogeneicantibody titers of 1:1000 and 1:300, respectively. The titers representthe dilution of the sera that gave a half maximal absorbance on ELISAplates coated with the respective polypeptides. Sera were pooled fromtwo rabbits, and the above titers represent the mean value. Onlyanti-sera to SEQ ID NO:7 cross-reacted with recombinant human CETP. Thereactivity of these anti-sera with rabbit CETP is under evaluation usingvarious immunological assays. Anti-polypeptide-specific IgG has beenpurified from the post-immune sera and its inhibitory property on humanrecombinant CETP is being assayed.

[0139] B. Immunization Strategy 2 (Purified Protein DerivativeConjugates)

[0140] Five of the above six rabbit CETP-polypeptide immunogens (SEQ IDNOs:2, 3, 4, 6 and 7) were coupled to tuberculin purified proteinderivative (PPD) according to the teachings of P. J. Lachmann et al., in1986 Synthetic Peptides As Antigens, (Ciba Foundation Symposium 119),25-40 (1986) and P. Dawson et al., J. Bio. Chem., 264:16798-16803 (1989)to form a conjugate. The tuberculin PPD (Statens Serum Inst.,Copenhagen, Denmark) was used as an exogenous antigenic carrier toenhance the immunogenicity of rabbit CETP-derived polypeptides. Thepolypeptide-PPD conjugate in PBS was emulsified with CFA as describedfor immunization strategy 1. One ml of 0.5 mg/ml polypeptide conjugatedto PPD was emulsified with approximately 1 ml CFA. A second 1 mlPPD-conjugate was frozen for next booster immunization.

[0141] On Day 1, rabbits were immunized with 1 ml of final emulsion inat least 10 sites sub-cutaneously on back of the rabbit. Thepolypeptide-PPD CETP polypeptide immunogen dose contained 0.25 mg ofpolypeptide per rabbit. Three weeks later (on Day 21), the rabbits weregiven the booster immunization dose with the remaining 1 ml conjugatethawed and emulsified with IFA, as discussed before. Two weeks followingthe second immunization rabbits were bled to collect post-immune sera.

[0142] The PPD conjugation strategy resulted in antibodies to theimmunogenic polypeptides of SEQ ID NOs:2 and 6, with antibody titers of1:3200 and 1:400 respectively. The titers represent the dilution of thesera that gave a half maximal absorbance on peptide coated ELISA plates.Sera were pooled from two rabbits and represent the mean value. Only theantibodies to the immunogenic polypeptide of SEQ ID NO:2 cross-reactedwith recombinant human CETP. These results were unexpectedly goodinasmuch as P. J. Lachmann et al., supra, obtained substantially noanti-polypeptide antibodies in BCG-naive hosts as were these rabbits.Anti-PPD antibodies were detected in all groups of rabbits as expected.

[0143] Using ELISA, the anti-immunogenic polypeptide sera are being usedto evaluate their immuno-reactivity with natural rabbit CETP. Becausethe polypeptides of SEQ ID NOs:2, 6 and 7 were immunogenic and the twoanti-polypeptide antibodies against SEQ ID NOs:2 and 7 immunologicallycross-reacted with recombinant human CETP, the respective rabbits werefurther boosted with a third immunization dose either with the MAPconstructs or PPD constructs emulsified with IFA.

Example 2 Immunization of Outbred Rabbits With CETP-Based Antigen

[0144] This study utilized 30 New Zealand white rabbits in three groupswith 10 rabbits per group. Three immunogens were utilized in this study:(1) Recombinant human CETP, (2) the carboxy-terminal 26 amino acidresidues of rabbit CETP (SEQ ID NO:50), and (3) a control immunogenwhose amino acid residue sequence was unrelated to that of CETP.

[0145] Pre-immune sera were collected before immunization with therespective immunogens. The purpose of this study was to illustrate thatthe above CETP immunogens would induce anti-CETP-specific (autogeneicanti-CETP) antibodies in rabbits, and that the autogeneic antibodiesgenerated against CETP bind to (immunoreact with) the endogenous rabbitCETP, and thus lessen the transfer of cholesteryl esters from HDLparticles and raise the level of HDL in the hosts.

[0146] The above immunogens were emulsified in CFA. Each rabbit received500 μg of one of the immunogens emulsified in CFA immunized bysub-cutaneous route. Seven weeks later the first bleed post-immune serawere collected.

[0147] ELISA was employed to titrate the antibodies. ELISA plates werecoated (40 ng/well) with the recombinant human CETP.

[0148] The rabbits immunized with recombinant human CETP exhibited aprimary immune response against human CETP. All the ten rabbitsresponded well to the recombinant human CETP (rhCETP). The specific IgGantibody titer was >1:1000. However, the group of 10 rabbits immunizedwith the rabbit CETP carboxy-terminal polypeptide-thyroglobulinconjugate (CETP-TH) did not exhibit a primary antibody response. Thecontrol rabbit sera had no detectable levels of anti-CETP antibodies.The rabbits were boosted with each respective antigen to further studyimmunogenicity.

[0149] The results of this study on the elevation of HDL particleconcentration in the blood (plasma) of the host mammals (mean±.S.D.) areshown in Table 1, below, for those first-immune sera. TABLE 1 HDL LevelsIn Immunized Animals (mg/dl) Avg.³ Immunogen HDL S.D.⁴ P⁵ Control 23.893.92 — rhCETP¹ 26.59 4.41 0.17 CETP-TH² 26.14 6.93 0.38

[0150] As can be seen from those results, an increase in HDL particleconcentration was found after administration of each of the CETPimmunogens. There was a relatively large scatter in the data.Nevertheless, an approximately 10 percent increase in the HDLcholesterol level was observed with each CETP immunogen as compared withthe control, with the recombinant human CETP immunogen providing itsincrease at a confidence level of greater than 80 percent (p=0.17) usinga Student's T test to analyze the results.

Example 3 Construction of E. coli Expression Vectors EncodingHBcAg/CETP/HBcAg Fusion Proteins

[0151] A. PCR amplification of HBcAg

[0152] Plasmid pFS14, a derivative of expression vector pKK223(Pharmacia), encodes HEcAg (subtype ayw) [Schödel et al., Infect. Immun.57:1347 (1989)]. PCR primer A, below, is designed to amplify the 5′ endof HBcAg and place an NcoI (C′C.ATG,G) site in the correct reading frameat the natural ATG start codon. In each of the sequences shownhereinafter, only the coding strand is shown, and bases removed aftercleavage by restriction enzymes are shown in lower case. Primer A:5′ gatccCATGGACATCGACCCTTATAAAGAATTTG SEQ ID NO:44 G 3′

[0153] Primer Z, below, is designed to amplify the 3′ end of HBcAg andplace a TAA stop codon and a HindIII (A′AGCT,T) site following aminoacid 183 (Cys). Primer Z: 5′ gatcaAGCTTTTAACATTGAGATTCCCGAGATTG SEQ IDNO:45 AGATCTTCTG 3′

[0154] A DNA fragment encoding the full-length HBcAg with modified 5′and 3′ ends is amplified using plasmid pFS14 DNA as a template in thepresence of primer A and primer Z under the standard polymerase chainreaction conditions recommended by the manufacturer of the GeneAmp PCRreagent kit (Perkin Elmer Cetus, Norwalk, Conn.).

[0155] The amplified DNA is then cleaved with NcoI and HindIII, andfractionated by size on an agarose gel. Full-length HBcAg DNA ispurified from a gel slice using a QIAQUICK™ gel extraction kit (QIAGEN,Chatsworth, Calif.).

[0156] B. Insertion of HBcAg into pProEx1

[0157] pProEx1, an E. coli expression vector (Life Technologies,Rockville, Md.), is also cleaved with NcoI and HindIII and gel-purified.The amplified DNA and pProEx1 DNAs are ligated under standard conditionsusing T4 DNA ligase and transformed into chemically-competent E. coliDH10B cells (Life Technologies) using protocols supplied by the vendorto form plasmid ProEx1-AZ. The transformation mixture is spread on LBagar plates containing 100 μg/ml ampicillin and incubated overnight at(about 18 hours) at 37° C. Colonies harboring ampicillin-resistantplasmids are purified by restreaking on fresh LB agar plates containingampicillin, and minipreps of plasmid DNA are prepared using WIZARD™ 373DNA purification kits (Promega, Inc., Madison, Wis.). Plasmidscontaining the HBcAg fragment inserted into the NcoI and HindIII sitesof pProEx1 are characterized by restriction mapping and sequenceanalysis across the inserted region.

[0158] Plasmid pProEx1-AZ is then modified to insert a polylinkerbetween the nucleotides that encode amino acid residues 70-75 of HBcAg.

[0159] Primer B is designed to insert an XhoI site (C′TCGA) and an EcoRIsite (G′AATT,C) site following position 206 of SEQ ID NO:39. Primer Y isdesigned to insert an EcoRI site (G′AATT,C) site followed by a SpeI site(A′CTAG,T) before position 226 of SEQ ID NO:39. Primer Y:5′ gatcgAATTCACTAGTTGGAAGATCCAQCGTCTA SEQ ID NO:46 GAGACCTAGTAG 3′Primer B: 5′ gatcgAATTCCTCGAGCTAGAGTCATTAGTTCCC SEQ ID NO:47 CCCAGCA 3′

[0160] Plasmid pProEx1-AZ is then used as a template with primers A andB to amplify a segment of DNA (designated HBcAg-AB) encoding amino acidresidues 1-69 of HBcAg to generate a fragment that contains an NcoI siteat its 5′ end and an XhoI and a EcoRI site at its 3′ end. The sameplasmid is also used with primers Y and Z to amplify a segment of DNA(designated HBcAg-YZ) encoding amino acid residues 76-183 of HBcAg togenerate a fragment that contains EcoRI and SpeI sites at its 5′ end anda HindIII site at its 3′ end.

[0161] The PCR product from the reaction designed to produce plasmidHBcAg-AB is cleaved with NcoI and EcoRI and purified after agarose gelelectrophoresis. The PCR product from a second reaction designed toproduce plasmid HBcAg-YZ is cleaved with EcoRI and HindIII and purifiedafter agarose gel electrophoresis. The two gel-purified fragments arethen ligated in a triple ligation reaction to plasmid pProEx1 that hadbeen treated with NcoI and HindIII and purified after agarose gelelectrophoresis. The desired ligated plasmid, pProEx1-AB-YZ, is obtainedby screening ampicillin-resistant colonies for plasmids that have thepredicted structure by restriction analysis, and is confirmed by DNAsequencing across the whole HBcAg region, particularly the A, BY, and Zjunctions.

[0162] C. Cloning Of CETP Segment Encoding SEQ ID NO:29

[0163] A stably transformed CHO cell line transfected with human CETPcDNA [Wang et al., J. Biol. Chem., 270:612-618 (1995); Wang et al., J.Biol. Chem., 267:17487-17490 (1992)] provides CETP cDNA that is used asa template to amplify a segment (nucleotides 1346 to 1431) of the CETPcoding sequence (SEQ ID NO: 1) that encodes the human peptide (SEQ IDNO:29; ArgAspGlyPheLeuLeuLeuGlnMetAspPheGlyPheProGluHisLeuLeuValAspPheLeuGlnSerLeuSer) that is bound by the antibody TP2; T. L.Swenson et al., J. Biol. Chem., 264:14318-14326 (1989).

[0164] Primer C, below, is designed to amplify a region from justupstream from the natural XhoI site at position 1346. Primer X, below,is designed to amplify a region at the 3′ end of the CETP gene, removingthe TAG codon and replacing it with an Eco47III site (AGC|GCT) followedby an EcoRI site (G′AATT,C). Primer C:5′ GATTATCACTCGAGATGGCTTCCTGCTGCTGCAG SEQ ID NO:48 3′ Primer X:5′ gatcgAATTCAGCGCTCAAGCTCTGGAGGAAAT SEQ ID NO:49 CCACCAG 3′

[0165] The CETP cDNA is then used as a template with primers C and X toamplify a segment of DNA (designated pCETP-CX) encoding amino acidresidues 461-476 of CETP, that contains an XhoI site near its 5′ end andan Eco47III and EcoRI site at its 3′ end. This segment, CETP-CX, is thencleaved with XhoI and EcoR47III, and gel-purified. Plasmid pProEx1-AB-YZis digested with SpeI and treated with T4 DNA polymerase to remove the4-base 5′ overhangs and generate blunt ends. [See, J. Sambrook et al.,Molecular Cloning, 2nd ed., Cold Spring Harbor Press, Cold Spring HarborN.Y., 8-23 (1989).]

[0166] The resulting plasmid is then treated with XhoI, gel-purified,and ligated to the segment CETP-CX that has an XhoI site at one end anda blunt end resulting from cleavage with Eco47III at the other end. Theresulting plasmid, designated pProEx1-ABC-XYZ, is characterized byrestriction analysis and by sequencing to confirm that it containssequences encoding amino acid residues 461-476 of CETP replacingsequences that encoded amino acid residues 70-75 of HBcAg in the vectorpProEx1-AZ.

Example 4 Expression of HBcAg/CETP/HbcAg Fusion Proteins In E. coli

[0167] The pProEx1 vector is designed for the expression of foreignproteins in E. coli. This vector contains a gene conferring resistanceto ampicillin and a pBR322 origin of replication for propagation in E.coli. It also has a multiple cloning site flanked by a 6 histidinesequence (6×His) and the recognition sequence for rTEV protease. Thissite allows for the removal of the 6×His tag from a fusion protein afterpurification. The vector also has a Trc promoter and lacI^(q) genepermitting inducible gene expression withisopropyl-β-D-thiogalactopyranoside (IPTG). A procaryotic ribosomalbinding site is located upstream from the start of translation of the6×His tag. A unique NcoI site is located at the first codon of the 6×Histag. Plasmids pProEx1 and a control plasmid, pProEx1-CAT, are obtainedfrom Life Technologies.

[0168]E. coli DH10B strains individually harboring pProEx1, pProEx1-CAT,pProEx1-AZ, or pProEx1-ABC-XYZ are cultured overnight (about 18 hours),and used as inocula for cultures that are induced with IPTG underconditions recommended by the vendor. Cultures harboring plasmidpProEx1-AZ produce HBcAg and those harboring pProEx1-ABC-XYZ produce thedesired HBcAg/CETP/HBcAg fusion protein as particles. These proteinslack the 6×His tag present in the original pProEx1 vector because theHBcAg sequences are inserted at the NcoI site at the beginning of the6×tag. Cultures harboring pProEx1-CAT produce a protein that migrates onSDS-PAGE gels as expected for a His tagged CAT fusion protein.

Example 5 Expression of HBcAg/CETP/HBcAg Fusion Proteins InBaculovirus-Infected Insect Cells

[0169] Baculovirus-infected insect cells have been shown to express awide variety of recombinant proteins (V. A. Luckow, Insect CellExpression Technology, pp. 30 183-218, in Protein Engineering:Principles and Practice, J. L. Cleland et al. eds., Wiley-Liss, Inc,1996). Heterologous genes placed under the control of the polyhedrinpromoter of the Autographa californica nuclear polyhedrosis virus(AcNPV) are often expressed at high levels during the late stages ofinfection. In most cases, the recombinant proteins are appropriatelyprocessed and are functionally similar to their authentic counterparts.

[0170] Recombinant baculoviruses containing the chimericHBcAg/CETP/HBcAg gene are constructed using the baculovirus shuttlevector system (Luckow et al., J. Virol., 67:4566-4579, 1993) soldcommercially as the Bac-To-Bac™ baculovirus expression system (LifeTechnologies).

[0171] Briefly, pProEx1-ABC-XYZ is digested with NcoI, treated withKlenow enzyme to fill in the ends, and digested with HindIII to releasethe entire fragment encoding the HBcAg/CETP/HBcAg fusion protein. Thisfragment is inserted into a baculovirus donor plasmid, pFastBac1, thatis digested with BamHI, treated with Klenow enzyme, and digested withHindIII. The resulting plasmid has the sequences encoding the hybridHBcAg/CETP/HBcAg gene inserted downstream from the polyhedrin promoterof AcNPV. The mini-Tn7 segment containing thepolyhedrin/HBcAg/CETP/HBcAg expression cassette is then transposed to abaculovirus shuttle vector propagated in E. coli and colonies harboringcomposite (recombinant) vectors are identified by their color and analtered drug resistance patterns. Miniprep DNAs are prepared andtransfected into cultured Spodoptera frugiperda (fall armyworm) Sf9cells.

[0172] Stocks of recombinant viruses are prepared and expression of therecombinant protein is monitored by standard protocols (O'Reilly et al.,Baculovirus Expression Vectors: A Laboratory Manual, W. H. Freeman andCompany, New York, 1992; King, L. A., and Possee, R. D. The BaculovirusExpression System: A Laboratory Guide, Chapman & Hall, London, 1992).

Example 6 Expression of HBcAg/CETP/HBcAg Fusion Proteins In MammalianCells

[0173] The HBcAg/CETP/HBcAg fusion protein is expressed in mammaliancell culture using the BHK/VP16 expression system (Hippenmeyer et al.,Bio/Technology, 11:1037-1041, 1993). Briefly, theHbcAg/CETP/HbcAg-encoding sequence of the NcoI-HindIII fragment fromplasmid pProEx1-ABC-XYZ of Example 3 is isolated by gel electrophoresisand purified as before. The fragment is treated with Klenow polymeraseand all four nucleotide triphosphates to make the 5′ overhanging endsblunt.

[0174] The mammalian expression vector pMON3327 contains the SV40polyadenylation signal sequence in the BamHI site of plasmid pUC18, andis used as the basis for further plasmid construction. Ligation of theIE175 promoter of herpes simplex virus (HSV-1) upstream of the SV40polyadenylation signal sequence in vector pMON3327 provides mammalianexpression vector pMON3360B. The IE175 promoter is responsive to theHSV-1 VP-16 transactivator.

[0175] Expression vector pMON3360B is digested with BamHI and the 5′overhanging ends at the unique BamHI site are filled in using Klenowpolymerase. The vector sequences and the HBcAg/CETP/HBcAg sequences areligated overnight (about 18 hours) at 15° C. using T4 DNA ligase. Theligation mixture is transfected into competent E. coli and selected forampicillin resistance. Plasmid DNA is isolated from the colonies andanalyzed by restriction analysis for proper orientation of theHBcAg/CETP/HBcAg sequences in the pMON3360B vector. A plasmid with thecorrect orientation is designated pMON3360B-HBcAg-CETP. PlasmidpMON3360B-HBcAg-CETP is purified using Promega Maxiprep™ protocols from400 ml cultures.

[0176] BHK/VP16 hamster kidney cells are plated at about 3×10⁵ cells per60 mm culture dish 24 hours before transfection in growth mediumconsisting of DMEM/5% fetal bovine sera (Life Technologies). Tenmicrograms of plasmid pMON336OB-HBcAg-CETP and 1 μg of plasmid pMON1118are transfected into the cells using LipofectAmine™ (Life Technologies)as recommended by the manufacturer. Two days after tranfection, thecells are treated with trypsin/EDTA (Life Technologies) and plated inten 100 mm dishes in growth medium containing hygromycin (Sigma). Inabout two weeks, surviving colonies are isolated using filter paper andexpanded and assayed for expression of the HBcAg/CETP/HBcAg fusionprotein.

Example 7 Construction of DNA Vaccine Vectors Capable of In VivoExpression of HBcAg/CETP/HBcAg Fusion Proteins

[0177] A. Construction of pcDNAI/Amp-HBcAg/CETP/HBcAg-1 andpcDNAI/Amp-HBcAg/CETP/HBcAg-2

[0178] Vector pcDNAI/Amp (Invitrogen Corp., San Diego, Calif.) is aderivative of vector pcDNAI and its parent vector pCDM8. All threevectors have the following features: Enhancer-promoter sequences fromthe immediate early gene of human cytomegalovirus (CMV) for high-levelconstitutive expression; SV40 poly(A) transcription termination and RNAprocessing signals to enhance mRNA stability, a versatile multiplecloning site to permit unidirectional or bidirectional cloning ofinserts; and a ColE1 origin of replication for growth in E. coli. VectorpcDNAI/Amp also contains bacteriophage T7 and SP6 promoters flanking anexpanded multiple cloning site, and an ampicillin resistance gene tofacilitate growth and selection in most E. coli strains. These vectorscan be used for high-level constitutive expression of recombinantproteins, including cytoplasmic proteins, transcription factors, viralproteins, cell surface receptors, and secreted proteins, in a variety ofmammalian cells.

[0179] Vector pcDNAI/Amp can be linearized at any of a variety ofpositions in the multiple cloning site downstream from the pCMV promoterfor insertion of heterologous genes. A blunt-ended DNA segment encodingHBcAg/CETP/HBcAg is prepared as described in Example 5 by digestingpProEx1-ABC-XYZ with NcoI and HindIII, and treating with Klenow enzymein the presence of all four nucleotide triphosphates to make the 5′overhanging ends blunt. This fragment is purified from an agarose gel.Vector pcDNAI/Amp is digested with EcoRV to leave blunt ends, treatedwith shrimp alkaline phosphatase to remove 5′ terminal phosphates, andligated to the purified fragment encoding HBcAg/CETP/HBcAg. Theresulting DNA is transformed into E. coli DH10B, and the resultingcolonies are screened for plasmids containing the HBcAg/CETP/HBcAginserted in the proper orientation downstream from the CMV promoter. Thedesired plasmid is designated pcDNAI/Amp-HBcAg/CETP/HBcAg-1.

[0180] A similar plasmid is also prepared by inserting the blunt-endedHBcAg/CETP/HBcAg into pcDNAI/Amp that is treated with BamHI, XbaI, andKlenow enzyme, to fill in the ends, to generatepcDNAI/Amp-HBcAg/CETP/HBcAg-2. This plasmid differs frompcDNAI/Amp-HBcAg/CETP/HBcAg-1 by removal of the large central portion ofthe multiple cloning site flanked by the CMV promoter and the SV40poly(A) regions.

[0181] B. Construction of pCMV-SPORT-HBcAg/CETP/HBcAg

[0182] Plasmid pCMV-SPORT-β-gal (Life Technologies, Rockville, Md.)contains the E. coli β-galactosidase gene cloned as a NotI fragment intopCMV-SPORT1. These vectors contain a CMV promoter, an SV40 poly(A) site,an ampicillin resistance gene, and an E. coli plasmid origin ofreplication. Plasmid pCMV-SPORT-β-gal is commonly used as a reportervector to monitor transfection efficiency. The plasmid PCMV-SPORTHBcAg/CETP/HBcAg is prepared using similar procedures describe above insubsection A. Plasmid pCMV-SPORT-β-gal is linearized with NotI, treatedwith Klenow to fill in the ends, and with shrimp alkaline phosphatase toremove 5′ terminal phosphates. The linearized, phosphatase-treatedvector is then ligated to the blunt end linear fragment from aboveencoding HBcAg/CETP/HBcAg. The ligation mixtures are transformed into E.coli DH10B, and the resulting plasmids screened for the properorientation of the insert into the vector.

[0183] Plasmids pCMV-SPORT-β-gal and pGreen Lantern-1 (LifeTechnologies, Rockville, Md.) both contain reporter genes under thecontrol of the CMV promoter and are suitable as control vectors for usein mammalian transfection studies. Expression of β-galactosidaseactivity is easily monitored by in situ staining of prokaryotic oreukaryotic cells with the chromogenic substrate X-gal. Plasmid pGreenLantern-1 contains a mutated form of the gene for Green FluorescentProtein (GFP) from Aequorea victoria jellyfish. GFP requires nosubstrates for visualization, and can be monitored in living as well asfixed cells, and in whole animals, by fluorescence microscopy using FITCfilters.

Example 8 Preparation of Purified DNA Template

[0184] Briefly, cultures of E. coli harboring plasmids are grown up inTB or LB media. [See, J. Sambrook et al., Molecular Cloning, 2nd ed.,Cold Spring Harbor Press, Cold Spring Harbor N.Y., 8-23 (1989).] PlasmidDNA is released from pelleted cells by an alkaline lysis method usingthe reagents supplied in a commercial anion exchange purification kit(Qiagen GmbH, Hilden, Germany). The recovered lysate is purified onP-2500 columns (Qiagen), precipitated with isopropanol, solubilized inTris-EDTA, reprecipitated in NaCl ethanol, and finally resuspended insterile endotoxin-free phosphate buffered saline (PBS; Sigma Chemical,St. Louis, Mo.). Plasmid DNA is stored at −20 degrees C. The bulkproduct is tested for the presence of sterility and the presence ofcontaminating DNA, RNA, protein, and endotoxin according to establishedprotocols [reviewed in M. Marquet et al., BioPharm, 10(5): 42-50(1997)].

Example 9 In Vivo Expression of DNA Encoding HBcAg/CETP/HBcAg FusionProteins Injected Directly into the Muscles of Mice

[0185] Endotoxin-free samples of plasmids pcDNAI/Amp-HBcAg/CETP/HBcAg-1and -2, pCMV-SPORT-HBcAg/CETP/HBcAg, pCMV-SPORT-β-gal, and pGreenLantern-1 are prepared as described in the preceding examples. Thequadriceps muscles of mice are injected with 100 μg of one of theplasmids listed above, and the muscle tissue at the site of injection isassayed for activity of the β-gal or GFP reporter gene or expression ofthe HBcAg/CETP/HBcAg fusion protein after a suitable maintenance time.

[0186] A. Light microscopy

[0187] The quadriceps muscles of mice immunized by to the plasmid DNAinjections, are removed in their entirety, cross-sectioned, andhistochemically-stained with X-gal for β-gal activity [Wolff et al.,Science, 247:1465-1468, 1990]. Only those tissues exposed topCMV-SPORT-β-gal produce the insoluble precipitate that is the indigoblue product resulting from cleavage of X-gal by the expressedβ-galactosidase protein. Similarly, samples expressing GFP when viewedby light microscopy using FITC filters, show a dramatic fluorescentsignal with an excitation peak of 490 nm. [R. Heim, et al., Nature, 373:663 (1995).] Samples expressing the fusion protein do not fluoresceunder these conditions nor react with X-gal.

[0188] B. Immunofluorescence

[0189] Muscle samples expressing the HBcAg/CETP/HBcAg fusion protein aredetected by immunofluorescence with primary antibodies directed againstHBcAg or CETP, and any of a number of well-known secondary antibodies(e.g. FITC- or Rhodamine-conjugated secondary antibodies). Establishedimmunofluorescence protocols are widely known in the art, and many ofthe reagents and methods are available from commercial sources.

[0190] C. Immunoblotting

[0191] Muscle extracts are prepared by mincing quadriceps tissue in amicrocentrifuge tube containing lysis buffer (20 mM Tris, pH 7.4, 2 MMMgCl₂, and 0.1% Triton X-100) and grinding with a plastic pestle untilhomogenized. Protein samples are analyzed on SDS-PAGE gels andelectrophoretically-transferred to Immobilon-P or nitrocellulosemembranes, using standard protocols. [See, J. Sambrook et al., MolecularCloning, 2nd ed., Cold Spring Harbor Press, Cold Spring Harbor N.Y.,8-23 (1989).]. Primary antibodies directed against β-galactosidase, GFP,HBcAg, and CETP obtained from commercial sources are used to detect theexpression products resulting from injection of plasmid DNAs into themuscle tissue. Many secondary antibodies are available from a widevariety of commercial sources that are chemically conjugated to areporter enzymes. Commercially available examples include alkalinephosphatase-conjugated anti-rabbit, anti-mouse, or anti-human IgG.Secondary antibodies chemically conjugated to horse radish peroxidase orto β-galactosidase are also widely available. Muscle extracts expressingGFP are detected with anti-GFP antibodies, those expressing β-galdetected with anti-β-gal antibodies, and those expressing theHBcAg/CETP/HBcAg fusion protein are detected with antibodies directedagainst HBcAg or CETP.

[0192] D. PCR analysis

[0193] Muscle extracts are also analyzed to detect presence of theinjected plasmid DNAs by polymerase chain reaction techniques. Briefly,muscle tissues taken from the site of injection are homogenized in lysisbuffer, template DNA is prepared, and used in reactions containingappropriate mixes of primers and a thermostable polymerase. Primerslying within the ampicillin resistance gene, common to all vectors,amplify a DNA fragment in all tissues that take up and maintain theinjected plasmid DNA. Primer sets specific for each gene, GFP, β-gal,HBcAg, and CETP, amplify uniquely-sized fragments, if the plasmid insertis intact within plasmids maintained within the cells. PCR can be usedto monitor the long term stability of the plasmid within muscle tissuesand aid in transcription and expression studies, if expression levelsmonitored by immunoblotting or microscopic techniques are low ornonexistent. PCR can also be used to determine whether the plasmidsintegrate into the host chromosome or are lost by passive diffusion.

[0194] E. Antibody Production

[0195] Mice immunized as discussed above produce antibodies thatimmunoreact with CETP in ELISA assays as discussed before. Similarlyimmunized rabbits also produce anti-CETP antibodies, which antibodiescause a lessening in the transfer of cholesteryl esters from HDL, andalso an increase in the HDL particle concentration in blood plasma.

Example 10 In Vivo Expression of DNA Encoding Rabbit CETP InjectedDirectly into the Muscles of Mice

[0196] A. PCR Amplification of Rabbit CETP

[0197] Rabbit CETP cDNA (SEQ ID NO: 27) [Nagashima et al., J. LipidRes., 29:1643-1649 (1988) or Kotake et al., Ibid, 37:599-605 (1996)] isobtained as described. PCR Primer N, below is designed to amplify the 5′end of rabbit CETP and place an NotI (GC′GGCC,GC) and an NcoI (C′CATG,G)site in the correct reading frame at the natural ATG start condonimmediately preceding the GCC condon at position +1 in SEQ ID NO:27. Ineach of the sequences shown below, only the coding strand is shown, withthe bases removed after cleavage by restriction enzymes not being shown,and synthetic sequences being shown in lower case. Primer N:5′ ggccgcccatgGCCTGTCCCAAAGGCGCCTCCTA (SEQ ID CGAGGCT 3′ NO:51)

[0198] Primer M, below, is designed to amplify the 3′ end of the rabbitCETP and place a TAA stop condon, a HindIII (A′ACGT,T) and NotI site(GC′GGCC,GC) following the amino acid residue 497 (Ser) immediatelypreceding the TAG stop codon ending at position +1494 of SEQ ID NO:27Primer M 5′ ggccgcacgttttaCTAGCTCAGGCTCTGCAGGA (SEQ ID AATCCACCAGCAGGTGNO:52)

[0199] A DNA fragment encoding the full-length rabbit CETP with modified5′ and 3′ ends is amplified using the above cDNA as a template in thepresence of primer M and primer N under the standard polymerase chainreaction conditions recommended by the manufacturer of the GeneAmp PCRreagent kit (Perkin Elmer Cetus, Norwalk, Conn.). The amplified DNA isthen cleaved with NotI and fractionated by size on an agarose gel.Full-length rabbit CETP DNA is purified as described above.

[0200] B. Insertion of Rabbit CETP DNA into pGEM-5Zf(+), pCR II, andpProEx1

[0201] The full-length rabbit CETP with NotI-compatible ends is insertedinto a plasmid cloning vector, such as pGEM™-5Zf(+) (PromegaCorporation, Madison, Wis.), which has a unique NotI site in thepolylinker region within the lacZalpha peptide region. Recombinants areselected by blue/white color screening as recommended by the vendor. Analternate procedure is to clone the PCR product containing thefull-length rabbit CETP directly into a TA cloning vector, such aspCR{fourth root} II (Invitrogen Corporation, San Diego, Calif.). DNAsequencing is carried out to confirm the identity of the nucleotidesequence encoding the rabbit CETP. In either case, the full-lengthrabbit CETP DNA can be released from the vector as a fragment withNotI-compatible ends, or as an NcoI-HindIII fragment.

[0202] C. Expression of Rabbit CETP in E. coli, Insect Cells, andMammalian Cells

[0203] The rabbit CETP is inserted into pProEx1 as an NcoI-HindIIIfragment as described above in Examples 3B and 3C for the DNA encodingthe HbcAg/CETP/HbcAg chimeric fusion protein. Expression of the rabbitCETP protein in E. coli is also carried out as described above inExample 4. The rabbit CETP protein is then purified from E. coli, toconfirm the biological activity of the isolated protein.

[0204] The DNA encoding rabbit CETP is purified and inserted intoplasmid pFastBac1 as described in Example 5. Recombinant baculovirusesare generated and used to express rabbit CETP in infected insect cells.

[0205] The DNA encoding rabbit CETP is purified and inserted intopMON3327 as described in Example 6. BHK/VP16 cells are transfected withthe resulting plasmid and surviving colonies are expanded and assayedfor expression of the rabbit CETP.

[0206] D. Insertion of the Rabbit CETP gene into DNA Vaccine Vectors

[0207] The rabbit CETP gene (rCETP) is inserted as a blunt-endedfragment into vector pcDNAI/Amp as described in Example 7A. The rabbitCETP gene is also inserted into pCMV-SPORT as an NotI fragment asdescribed in Example 7B. Both plasmids direct expression of rabbit CETPin mammalian cells under the control of the CMV promoter.

[0208] Mammalian cells are transfected with pcDNAI/Amp-rCETP orpCMV-SPORT-rCETP. Expression of rabbit CETP is assayed byimmunofluorescence techniques as described in Example 9B and byimmunoblotting techniques, as described in Example 9C.

[0209] Once rabbit CETP is detectable and expression levels are suitablyhigh in transfected mammalian cell lines to provide assurance ofexpression, injection of pcDNAI/Amp-rCETP or pCMV-SPORT-rCETP intomuscle tissues of animals containing CETP in their blood, such as Guineapigs for example, is carried out and monitored as described in Examples9A-E.

[0210] The foregoing description and the examples are intended asillustrative and are not to be taken as limiting. Still other variationswithin the spirit and scope of this invention are possible and willreadily present themselves to those skilled in the art.

0 SEQUENCE LISTING (1) GENERAL INFORMATION: (iii) NUMBER OF SEQUENCES:52 (2) INFORMATION FOR SEQ ID NO:1: (i) SEQUENCE CHARACTERISTICS: (A)LENGTH: 1431 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single(D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (viii) POSITIONIN GENOME: (C) UNITS: bp (x) PUBLICATION INFORMATION: (A) AUTHORS:Drayna, Dennis Jarnagin, Alisha Stephens McLean, John Henzel, WilliamKohr, William Fielding, Christopher Lawn, Richard (B) TITLE: Cloning andsequencing of human cholesteryl ester transfer protein cDNA (C) JOURNAL:Nature (D) VOLUME: 327 (F) PAGES: 632-634 (G) DATE: June 18-1987 (xi)SEQUENCE DESCRIPTION: SEQ ID NO:1: TGCTCCAAAG GCACCTCGCA CGAGGCAGGCATCGTGTGCC GCATCACCAA GCCTGCCCTC 60 CTGGTGTTGA ACCACGAGAC TGCCAAGGTCATCCAGACCG CCTTCCAGCG AGCCAGCTAC 120 CCAGATATCA CGGGCGAGAA GGCCATGATGCTCCTTGGCC AAGTCAAGTA TGGGTTGCAC 180 AACATCCAGA TCAGCCACTT GTCCATCGCCAGCAGCCAGG TGGAGCTGGT GGAAGCCAAG 240 TCCATTGATG TCTCCATTCA GAACGTGTCTGTGGTCTTCA AGGGGACCCT GAAGTATGGC 300 TACACCACTG CCTGGTGGCT GGGTATTGATCAGTCCATTG ACTTCGAGAT CGACTCTGCC 360 ATTGACCTCC AGATCAACAC ACAGCTGACCTGTGACTCTG GTAGAGTGCG GACCGATGCC 420 CCTGACTGCT ACCTGTCTTT CCATAAGCTGCTCCTGCATC TCCAAGGGGA GCGAGAGCCT 480 GGGTGGATCA AGCAGCTGTT CACAAATTTCATCTCCTTCA CCCTGAAGCT GGTCCTGAAG 540 GGACAGATCT GCAAAGAGAT CAACGTCATCTCTAACATCA TGGCCGATTT TGTCCAGACA 600 AGGGCTGCCA GCATCCTTTC AGATGGAGACATTGGGGTGG ACATTTCCCT GACAGGTGAT 660 CCCGTCATCA CAGCCTCCTA CCTGGAGTCCCATCACAAGG GTCATTTCAT CTACAAGAAT 720 GTCTCAGAGG ACCTCCCCCT CCCCACCTTCTCGCCCACAC TGCTGGGGGA CTCCCGCATG 780 CTGTACTTCT GGTTCTCTGA GCGAGTCTTCCACTCGCTGG CCAAGGTAGC TTTCCAGGAT 840 GGCCGCCTCA TGCTCAGCCT GATGGGAGACGAGTTCAAGG CAGTGCTGGA GACCTGGGGC 900 TTCAACACCA ACCAGGAAAT CTTCCAAGAGGTTGTCGGCG GCTTCCCCAG CCAGGCCCAA 960 GTCACCGTCC ACTGCCTCAA GATGCCCAAGATCTCCTGCC AAAACAAGGG AGTCGTGGTC 1020 AATTCTTCAG TGATGGTGAA ATTCCTCTTTCCACGCCCAG ACCAGCAACA TTCTGTAGCT 1080 TACACATTTG AAGAGGATAT CGTGACTACCGTCCAGGCCT CCTATTCTAA GAAAAAGCTC 1140 TTCTTAAGCC TCTTGGATTT CCAGATTACACCAAAGACTG TTTCCAACTT GACTGAGAGC 1200 AGCTCCGAGT CCATCCAGAG CTTCCTGCAGTCAATGATCA CCGCTGTGGG CATCCCTGAG 1260 GTCATGTCTC GGCTCGAGGT AGTGTTTACAGCCCTCATGA ACAGCAAAGG CGTGAGCCTC 1320 TTCGACATCA TCAACCCTGA GATTATCACTCGAGATGGCT TCCTGCTGCT GCAGATGGAC 1380 TTTGGCTTCC CTGAGCACCT GCTGGTGGATTTCCTCCAGA GCTTGAGCTA G 1431 (2) INFORMATION FOR SEQ ID NO:2: (i)SEQUENCE CHARACTERISTICS: (A) LENGTH: 20 amino acids (B) TYPE: aminoacid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE:peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2: Glu Ile Phe Gln Glu LeuSer Arg Gly Leu Pro Thr Gly Gln Ala Gln 1 5 10 15 Val Ala Val His 20 (2)INFORMATION FOR SEQ ID NO:3: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH:20 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: single (D)TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION:SEQ ID NO:3: Val Ala Val Thr Phe Arg Phe Pro Arg Pro Asp Gly Arg Glu AlaVal 1 5 10 15 Ala Tyr Arg Phe 20 (2) INFORMATION FOR SEQ ID NO:4: (i)SEQUENCE CHARACTERISTICS: (A) LENGTH: 22 amino acids (B) TYPE: aminoacid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE:peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4: Leu Leu Leu Gln Met AspPhe Gly Phe Pro Lys His Leu Leu Val Asp 1 5 10 15 Phe Leu Gln Ser LeuSer 20 (2) INFORMATION FOR SEQ ID NO:5: (i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 20 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: single(D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (xi) SEQUENCEDESCRIPTION: SEQ ID NO:5: Thr Thr Val Gln Ala Ser Tyr Ser Gln Lys LysLeu Phe Leu His Leu 1 5 10 15 Leu Asp Phe Gln 20 (2) INFORMATION FOR SEQID NO:6: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 20 amino acids (B)TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii)MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6: Leu LeuLeu His Leu Gln Gly Glu Arg Glu Pro Gly Trp Leu Lys Gln 1 5 10 15 LeuPhe Thr Asn 20 (2) INFORMATION FOR SEQ ID NO:7: (i) SEQUENCECHARACTERISTICS: (A) LENGTH: 20 amino acids (B) TYPE: amino acid (C)STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide(xi) SEQUENCE DESCRIPTION: SEQ ID NO:7: Asp Val Ser Gly Glu Arg Ala ValMet Leu Leu Gly Arg Val Lys Tyr 1 5 10 15 Gly Leu His Asn 20 (2)INFORMATION FOR SEQ ID NO:8: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH:20 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: single (D)TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION:SEQ ID NO:8: Gln Glu Ile Phe Gln Glu Val Val Gly Gly Phe Pro Ser Gln AlaGln 1 5 10 15 Val Thr Val His 20 (2) INFORMATION FOR SEQ ID NO:9: (i)SEQUENCE CHARACTERISTICS: (A) LENGTH: 20 amino acids (B) TYPE: aminoacid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE:peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO:9: Val Met Val Lys Phe LeuPhe Pro Arg Pro Asp Gln Gln His Ser Val 1 5 10 15 Ala Tyr Thr Phe 20 (2)INFORMATION FOR SEQ ID NO:10: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH:22 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: single (D)TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION:SEQ ID NO:10: Leu Leu Leu Gln Met Asp Phe Gly Phe Pro Glu His Leu LeuVal Asp 1 5 10 15 Phe Leu Gln Ser Leu Ser 20 (2) INFORMATION FOR SEQ IDNO:11: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 20 amino acids (B)TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii)MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO:11: Thr ThrVal Gln Ala Ser Tyr Ser Lys Lys Lys Leu Phe Leu Ser Leu 1 5 10 15 LeuAsp Phe Gln 20 (2) INFORMATION FOR SEQ ID NO:12: (i) SEQUENCECHARACTERISTICS: (A) LENGTH: 20 amino acids (B) TYPE: amino acid (C)STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide(xi) SEQUENCE DESCRIPTION: SEQ ID NO:12: Leu Leu Leu His Leu Gln Gly GluArg Glu Pro Gly Trp Ile Lys Gln 1 5 10 15 Leu Phe Thr Asn 20 (2)INFORMATION FOR SEQ ID NO:13: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH:20 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: single (D)TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION:SEQ ID NO:13: Asp Ile Thr Gly Glu Lys Ala Met Met Leu Leu Gly Gln ValLys Tyr 1 5 10 15 Gly Leu His Asn 20 (2) INFORMATION FOR SEQ ID NO:14:(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 63 base pairs (B) TYPE:nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULETYPE: DNA (genomic) (viii) POSITION IN GENOME: (C) UNITS: bp (xi)SEQUENCE DESCRIPTION: SEQ ID NO:14: CAGGAAATCT TCCAGGAGCT TTCCAGAGGCCTTCCCACCG GCCAGGCCCA GGTAGCCGTC 60 CAC 63 (2) INFORMATION FOR SEQ IDNO:15: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 60 base pairs (B) TYPE:nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULETYPE: DNA (genomic) (viii) POSITION IN GENOME: (C) UNITS: bp (xi)SEQUENCE DESCRIPTION: SEQ ID NO:15: GTCGCCGTGA CGTTCCGCTT CCCCCGCCCAGATGGCCGAG AAGCTGTGGC CTACAGGTTT 60 (2) INFORMATION FOR SEQ ID NO:16:(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 66 base pairs (B) TYPE:nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULETYPE: DNA (genomic) (viii) POSITION IN GENOME: (C) UNITS: bp (xi)SEQUENCE DESCRIPTION: SEQ ID NO:16: CTGCTGCTGC AGATGGACTT CGGTTTTCCCAAGCACCTGC TGGTGGATTT CCTGCAGAGC 60 CTGAGC 66 (2) INFORMATION FOR SEQ IDNO:17: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 60 base pairs (B) TYPE:nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULETYPE: DNA (genomic) (viii) POSITION IN GENOME: (C) UNITS: bp (xi)SEQUENCE DESCRIPTION: SEQ ID NO:17: ACCACCGTCC AGGCCTCCTA CTCCCAGAAAAAGCTCTTCC TACACCTCTT GGATTTCCAG 60 (2) INFORMATION FOR SEQ ID NO:18:(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 60 base pairs (B) TYPE:nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULETYPE: DNA (genomic) (viii) POSITION IN GENOME: (C) UNITS: bp (xi)SEQUENCE DESCRIPTION: SEQ ID NO:18: CTGCTCCTGC ACCTCCAGGG GGAGCGCGAGCCGGGGTGGC TCAAGCAGCT CTTCACAAAC 60 (2) INFORMATION FOR SEQ ID NO:19:(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 60 base pairs (B) TYPE:nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULETYPE: DNA (genomic) (viii) POSITION IN GENOME: (C) UNITS: bp (xi)SEQUENCE DESCRIPTION: SEQ ID NO:19: GACGTCAGCG GCGAGAGGGC CGTGATGCTCCTCGGCCGGG TCAAGTACGG GCTGCACAAC 60 (2) INFORMATION FOR SEQ ID NO:20:(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 63 base pairs (B) TYPE:nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULETYPE: DNA (genomic) (viii) POSITION IN GENOME: (C) UNITS: bp (xi)SEQUENCE DESCRIPTION: SEQ ID NO:20: CAGGAAATCT TCCAAGAGGT TGTCGGCGGCTTCCCCAGCC AGGCCCAAGT CACCGTCCAC 60 TGC 63 (2) INFORMATION FOR SEQ IDNO:21: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 60 base pairs (B) TYPE:nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULETYPE: DNA (genomic) (viii) POSITION IN GENOME: (C) UNITS: bp (xi)SEQUENCE DESCRIPTION: SEQ ID NO:21: GTGATGGTGA AATTCCTCTT TCCACGCCCAGACCAGCAAC ATTCTGTAGC TTACACATTT 60 (2) INFORMATION FOR SEQ ID NO:22:(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 66 base pairs (B) TYPE:nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULETYPE: DNA (genomic) (viii) POSITION IN GENOME: (C) UNITS: bp (xi)SEQUENCE DESCRIPTION: SEQ ID NO:22: CTGCTGCTGC AGATGGACTT TGGCTTCCCTGAGCACCTGC TGGTGGATTT CCTCCAGAGC 60 TTGAGC 66 (2) INFORMATION FOR SEQ IDNO:23: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 60 base pairs (B) TYPE:nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULETYPE: DNA (genomic) (viii) POSITION IN GENOME: (C) UNITS: bp (xi)SEQUENCE DESCRIPTION: SEQ ID NO:23: ACTACCGTCC AGGCCTCCTA TTCTAAGAAAAAGCTCTTCT TAAGCCTCTT GGATTTCCAG 60 (2) INFORMATION FOR SEQ ID NO:24:(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 60 base pairs (B) TYPE:nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULETYPE: DNA (genomic) (viii) POSITION IN GENOME: (C) UNITS: bp (xi)SEQUENCE DESCRIPTION: SEQ ID NO:24: CTGCTCCTGC ATCTCCAAGG GGAGCGAGAGCCTGGGTGGA TCAAGCAGCT GTTCACAAAT 60 (2) INFORMATION FOR SEQ ID NO:25:(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 60 base pairs (B) TYPE:nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULETYPE: DNA (genomic) (viii) POSITION IN GENOME: (C) UNITS: bp (xi)SEQUENCE DESCRIPTION: SEQ ID NO:25: GATATCACGG GCGAGAAGGC CATGATGCTCCTTGGCCAAG TCAAGTATGG GTTGCACAAC 60 (2) INFORMATION FOR SEQ ID NO:26:(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 497 amino acids (B) TYPE:amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULETYPE: protein (x) PUBLICATION INFORMATION: (A) AUTHORS: Nagashima, M.McLean, J. W. Lawn, R. M. (B) TITLE: Cloning and mRNA tissuedistribution of rabbit cholesteryl ester transfer protein (C) JOURNAL:J. Lipid Res. (D) VOLUME: 29 (F) PAGES: 1643-1649 (G) DATE: 1988 (xi)SEQUENCE DESCRIPTION: SEQ ID NO:26: Ala Cys Pro Lys Gly Ala Ser Tyr GluAla Gly Ile Val Cys Arg Ile 1 5 10 15 Thr Lys Pro Ala Leu Leu Val LeuAsn Gln Glu Thr Ala Lys Val Val 20 25 30 Gln Thr Ala Phe Gln Arg Ala GlyTyr Pro Asp Val Ser Gly Glu Arg 35 40 45 Ala Val Met Leu Leu Gly Arg ValLys Tyr Gly Leu His Asn Leu Gln 50 55 60 Ile Ser His Leu Ser Ile Ala SerSer Gln Val Glu Leu Val Asp Ala 65 70 75 80 Lys Thr Ile Asp Val Ala IleGln Asn Val Ser Val Val Phe Lys Gly 85 90 95 Thr Leu Asn Tyr Ser Tyr ThrSer Ala Trp Gly Leu Gly Ile Asn Gln 100 105 110 Ser Val Asp Phe Glu IleAsp Ser Ala Ile Asp Leu Gln Ile Asn Thr 115 120 125 Glu Leu Thr Cys AspAla Gly Ser Val Arg Thr Asn Ala Pro Asp Cys 130 135 140 Tyr Leu Ala PheHis Lys Leu Leu Leu His Leu Gln Gly Glu Arg Glu 145 150 155 160 Pro GlyTrp Leu Lys Gln Leu Phe Thr Asn Phe Ile Ser Phe Thr Leu 165 170 175 LysLeu Ile Leu Lys Arg Gln Val Cys Asn Glu Ile Asn Thr Ile Ser 180 185 190Asn Ile Met Ala Asp Phe Val Gln Thr Arg Ala Ala Ser Ile Leu Ser 195 200205 Asp Gly Asp Ile Gly Val Asp Ile Ser Val Thr Gly Ala Pro Val Ile 210215 220 Thr Ala Thr Tyr Leu Glu Ser His His Lys Gly His Phe Thr His Lys225 230 235 240 Asn Val Ser Glu Ala Phe Pro Leu Arg Ala Phe Pro Pro GlyLeu Leu 245 250 255 Gly Asp Ser Arg Met Leu Tyr Phe Trp Phe Ser Asp GlnVal Leu Asn 260 265 270 Ser Leu Ala Arg Ala Ala Phe Gln Glu Gly Arg LeuVal Leu Ser Leu 275 280 285 Thr Gly Asp Glu Phe Lys Lys Val Leu Glu ThrGln Gly Phe Asp Thr 290 295 300 Asn Gln Glu Ile Phe Gln Glu Leu Ser ArgGly Leu Pro Thr Gly Gln 305 310 315 320 Ala Gln Val Ala Val His Cys LeuLys Val Pro Lys Ile Ser Cys Gln 325 330 335 Asn Arg Gly Val Val Val SerSer Ser Val Ala Val Thr Phe Arg Phe 340 345 350 Pro Arg Pro Asp Gly ArgGlu Ala Val Ala Tyr Arg Phe Glu Glu Asp 355 360 365 Ile Ile Thr Thr ValGln Ala Ser Tyr Ser Gln Lys Lys Leu Phe Leu 370 375 380 His Leu Leu AspPhe Gln Cys Val Pro Ala Ser Gly Arg Ala Gly Ser 385 390 395 400 Ser AlaAsn Leu Ser Val Ala Leu Arg Thr Glu Ala Lys Ala Val Ser 405 410 415 AsnLeu Thr Glu Ser Arg Ser Glu Ser Leu Gln Ser Ser Leu Arg Ser 420 425 430Leu Ile Ala Thr Val Gly Ile Pro Glu Val Met Ser Arg Leu Glu Val 435 440445 Ala Phe Thr Ala Leu Met Asn Ser Lys Gly Leu Asp Leu Phe Glu Ile 450455 460 Ile Asn Pro Glu Ile Ile Thr Leu Asp Gly Cys Leu Leu Leu Gln Met465 470 475 480 Asp Phe Gly Phe Pro Lys His Leu Leu Val Asp Phe Leu GlnSer Leu 485 490 495 Ser (2) INFORMATION FOR SEQ ID NO:27: (i) SEQUENCECHARACTERISTICS: (A) LENGTH: 1494 base pairs (B) TYPE: nucleic acid (C)STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA(genomic) (viii) POSITION IN GENOME: (C) UNITS: bp (x) PUBLICATIONINFORMATION: (A) AUTHORS: Nagashima, Mariko McLean, John W. Lawn,Richard M. (B) TITLE: Cloning and mRNA tissue distribution of rabbitcholesteryl ester transfer protein (C) JOURNAL: J. Lipid Res. (D)VOLUME: 29 (F) PAGES: 1643-1649 (G) DATE: 1988 (xi) SEQUENCEDESCRIPTION: SEQ ID NO:27: GCCTGTCCCA AAGGCGCCTC CTACGAGGCT GGCATCGTGTGTCGCATCAC CAAGCCCGCC 60 CTCTTGGTGT TGAACCAAGA GACGGCCAAG GTGGTCCAGACGGCCTTCCA GCGCGCCGGC 120 TATCCGGACG TCAGCGGCGA GAGGGCCGTG ATGCTCCTCGGCCGGGTCAA GTACGGGCTG 180 CACAACCTCC AGATCAGCCA CCTGTCCATC GCCAGCAGCCAGGTGGAGCT GGTGGACGCC 240 AAGACCATCG ACGTCGCCAT CCAGAACGTG TCCGTGGTCTTCAAGGGGAC CCTGAACTAC 300 AGCTACACGA GTGCCTGGGG GTTGGGCATC AATCAGTCTGTCGACTTCGA GATCGACTCT 360 GCCATTGACC TCCAGATCAA CACAGAGCTG ACCTGCGACGCTGGCAGTGT GCGCACCAAT 420 GCCCCCGACT GCTACCTGGC TTTCCATAAA CTGCTCCTGCACCTCCAGGG GGAGCGCGAG 480 CCGGGGTGGC TCAAGCAGCT CTTCACAAAC TTCATCTCCTTCACCCTGAA GCTGATTCTG 540 AAGCGACAGG TCTGCAATGA GATCAACACC ATCTCCAACATCATGGCTGA CTTTGTCCAG 600 ACGAGGGCCG CCAGCATCCT CTCAGATGGA GACATCGGGGTGGACATTTC CGTGACGGGG 660 GCCCCTGTCA TCACAGCCAC CTACCTGGAG TCCCATCACAAGGGTCACTT CACGCACAAG 720 AACGTCTCCG AGGCCTTCCC CCTCCGCGCC TTCCCGCCCGGTCTTCTGGG GGACTCCCGC 780 ATGCTCTACT TCTGGTTCTC CGATCAAGTG CTCAACTCCCTGGCCAGGGC CGCCTTCCAG 840 GAGGGCCGTC TCGTGCTCAG CCTGACAGGG GATGAGTTCAAGAAAGTGCT GGAGACCCAG 900 GGTTTCGACA CCAACCAGGA AATCTTCCAG GAGCTTTCCAGAGGCCTTCC CACCGGCCAG 960 GCCCAGGTAG CCGTCCACTG CCTTAAGGTG CCCAAGATCTCCTGCCAGAA CCGGGGTGTC 1020 GTGGTGTCTT CTTCCGTCGC CGTGACGTTC CGCTTCCCCCGCCCAGATGG CCGAGAAGCT 1080 GTGGCCTACA GGTTTGAGGA GGATATCATC ACCACCGTCCAGGCCTCCTA CTCCCAGAAA 1140 AAGCTCTTCC TACACCTCTT GGATTTCCAG TGCGTGCCGGCCAGCGGAAG GGCAGGCAGC 1200 TCAGCAAATC TCTCCGTGGC CCTCAGGACT GAGGCTAAGGCTGTTTCCAA CCTGACTGAG 1260 AGCCGCTCCG AGTCCCTGCA GAGCTCTCTC CGCTCCCTGATCGCCACGGT GGGCATCCCG 1320 GAGGTCATGT CTCGGCTCGA GGTGGCGTTC ACAGCCCTCATGAACAGCAA AGGCCTGGAC 1380 CTCTTCGAAA TCATCAACCC CGAGATTATC ACTCTCGATGGCTGCCTGCT GCTGCAGATG 1440 GACTTCGGTT TTCCCAAGCA CCTGCTGGTG GATTTCCTGCAGAGCCTGAG CTAG 1494 (2) INFORMATION FOR SEQ ID NO:28: (i) SEQUENCECHARACTERISTICS: (A) LENGTH: 476 amino acids (B) TYPE: amino acid (C)STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein(x) PUBLICATION INFORMATION: (A) AUTHORS: Drayna, Dennis Jarnagin,Alisha Stephens McLean, John Henzel, William Kohr, William Fielding,Christopher Lawn, Richard (B) TITLE: Cloning and sequencing of humancholesteryl ester transfer protein cDNA (C) JOURNAL: Nature (D) VOLUME:327 (F) PAGES: 632-634 (G) DATE: June 18-1987 (xi) SEQUENCE DESCRIPTION:SEQ ID NO:28: Cys Ser Lys Gly Thr Ser His Glu Ala Gly Ile Val Cys ArgIle Thr 1 5 10 15 Lys Pro Ala Leu Leu Val Leu Asn His Glu Thr Ala LysVal Ile Gln 20 25 30 Thr Ala Phe Gln Arg Ala Ser Tyr Pro Asp Ile Thr GlyGlu Lys Ala 35 40 45 Met Met Leu Leu Gly Gln Val Lys Tyr Gly Leu His AsnIle Gln Ile 50 55 60 Ser His Leu Ser Ile Ala Ser Ser Gln Val Glu Leu ValGlu Ala Lys 65 70 75 80 Ser Ile Asp Val Ser Ile Gln Asn Val Ser Val ValPhe Lys Gly Thr 85 90 95 Leu Lys Tyr Gly Tyr Thr Thr Ala Trp Trp Leu GlyIle Asp Gln Ser 100 105 110 Ile Asp Phe Glu Ile Asp Ser Ala Ile Asp LeuGln Ile Asn Thr Gln 115 120 125 Leu Thr Cys Asp Ser Gly Arg Val Arg ThrAsp Ala Pro Asp Cys Tyr 130 135 140 Leu Ser Phe His Lys Leu Leu Leu HisLeu Gln Gly Glu Arg Glu Pro 145 150 155 160 Gly Trp Ile Lys Gln Leu PheThr Asn Phe Ile Ser Phe Thr Leu Lys 165 170 175 Leu Val Leu Lys Gly GlnIle Cys Lys Glu Ile Asn Val Ile Ser Asn 180 185 190 Ile Met Ala Asp PheVal Gln Thr Arg Ala Ala Ser Ile Leu Ser Asp 195 200 205 Gly Asp Ile GlyVal Asp Ile Ser Leu Thr Gly Asp Pro Val Ile Thr 210 215 220 Ala Ser TyrLeu Glu Ser His His Lys Gly His Phe Ile Tyr Lys Asn 225 230 235 240 ValSer Glu Asp Leu Pro Leu Pro Thr Phe Ser Pro Thr Leu Leu Gly 245 250 255Asp Ser Arg Met Leu Tyr Phe Trp Phe Ser Glu Arg Val Phe His Ser 260 265270 Leu Ala Lys Val Ala Phe Gln Asp Gly Arg Leu Met Leu Ser Leu Met 275280 285 Gly Asp Glu Phe Lys Ala Val Leu Glu Thr Trp Gly Phe Asn Thr Asn290 295 300 Gln Glu Ile Phe Gln Glu Val Val Gly Gly Phe Pro Ser Gln AlaGln 305 310 315 320 Val Thr Val His Cys Leu Lys Met Pro Lys Ile Ser CysGln Asn Lys 325 330 335 Gly Val Val Val Asn Ser Ser Val Met Val Lys PheLeu Phe Pro Arg 340 345 350 Pro Asp Gln Gln His Ser Val Ala Tyr Tyr PheGlu Glu Asp Ile Val 355 360 365 Thr Thr Val Gln Ala Ser Tyr Ser Lys LysLys Leu Phe Leu Ser Leu 370 375 380 Leu Asp Phe Gln Ile Thr Pro Lys ThrVal Ser Asn Leu Thr Glu Ser 385 390 395 400 Ser Ser Glu Ser Ile Gln SerPhe Leu Gln Ser Met Ile Thr Ala Val 405 410 415 Gly Ile Pro Glu Val MetSer Arg Leu Glu Val Val Phe Thr Ala Leu 420 425 430 Met Asn Ser Lys GlyVal Ser Leu Phe Asp Ile Ile Asn Pro Glu Ile 435 440 445 Ile Thr Arg AspGly Phe Leu Leu Leu Gln Met Asp Phe Gly Phe Pro 450 455 460 Glu His LeuLeu Val Asp Phe Leu Gln Ser Leu Ser 465 470 475 (2) INFORMATION FOR SEQID NO:29: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 26 amino acids (B)TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii)MOLECULE TYPE: peptide (x) PUBLICATION INFORMATION: (A) AUTHORS:Swenson, T. L. et al., (C) JOURNAL: J. Biol. Chem. (D) VOLUME: 264 (F)PAGES: 14318-14326 (G) DATE: 1989 (xi) SEQUENCE DESCRIPTION: SEQ IDNO:29: Arg Asp Gly Phe Leu Leu Leu Gln Met Asp Phe Gly Phe Pro Glu His 15 10 15 Leu Leu Val Asp Phe Leu Gln Ser Leu Ser 20 25 (2) INFORMATIONFOR SEQ ID NO:30: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 493 aminoacids (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear(ii) MOLECULE TYPE: protein (x) PUBLICATION INFORMATION: (A) AUTHORS:Pape, Michael E. Rehberg, Edward F. Marotti, Keith R. Melchior, GeorgeW. (B) TITLE: Molecular Cloning, Sequence, and Expression of CynomolgusMonkey Cholesteryl Ester Transfer Protein (C) JOURNAL: Arteriosclerosisand Thrombosis (D) VOLUME: 11 (E) ISSUE: 6 (F) PAGES: 1759-1771 (G)DATE: Nov/Dec-1991 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:30: Met Leu AlaAla Thr Val Leu Thr Leu Ala Leu Leu Gly Asn Val His 1 5 10 15 Ala CysSer Lys Gly Thr Ser His Lys Ala Gly Ile Val Cys Arg Ile 20 25 30 Thr LysPro Ala Leu Leu Val Leu Asn Gln Glu Thr Ala Lys Val Ile 35 40 45 Gln SerAla Phe Gln Arg Ala Asn Tyr Pro Asn Ile Thr Gly Glu Lys 50 55 60 Ala MetMet Leu Leu Gly Gln Val Lys Tyr Gly Leu His Asn Ile Gln 65 70 75 80 IleSer His Leu Ser Ile Ala Ser Ser Arg Val Glu Leu Val Glu Ala 85 90 95 LysSer Ile Asp Val Ser Ile Gln Asn Val Ser Val Val Phe Lys Gly 100 105 110Thr Leu Lys Tyr Gly Tyr Thr Thr Ala Trp Gly Leu Gly Ile Asp Gln 115 120125 Ser Val Asp Phe Glu Ile Asp Ser Ala Ile Asp Leu Gln Ile Asn Thr 130135 140 Gln Leu Thr Cys Asp Ser Gly Arg Val Arg Thr Asp Ala Pro Asp Cys145 150 155 160 Tyr Leu Ser Phe His Lys Leu Leu Leu His Leu Gln Gly GluArg Glu 165 170 175 Pro Gly Trp Ile Lys Gln Leu Phe Thr Asn Phe Ile SerPhe Thr Leu 180 185 190 Lys Leu Val Leu Lys Gly Gln Ile Cys Lys Glu IleAsn Ile Ile Ser 195 200 205 Asn Ile Met Ala Asp Phe Val Gln Thr Arg AlaAla Ser Ile Leu Ser 210 215 220 Asp Gly Asp Ile Gly Val Asp Ile Ser LeuThr Gly Asp Pro Ile Ile 225 230 235 240 Thr Ala Ser Tyr Leu Glu Ser HisHis Lys Gly Tyr Phe Ile Tyr Lys 245 250 255 Asn Val Ser Glu Asp Leu ProLeu Pro Thr Phe Ser Pro Ala Leu Leu 260 265 270 Gly Asp Ser Arg Met LeuTyr Phe Trp Phe Ser Glu Gln Val Phe His 275 280 285 Ser Leu Ala Lys ValAla Phe Gln Asp Gly Arg Leu Thr Leu Ser Leu 290 295 300 Met Gly Asp GluPhe Lys Ala Val Leu Glu Thr Trp Gly Phe Asn Thr 305 310 315 320 Asn GlnGlu Ile Phe Gln Glu Val Val Gly Gly Phe Pro Ser Gln Ala 325 330 335 GlnVal Thr Val His Cys Leu Lys Met Pro Arg Ile Ser Cys Gln Asn 340 345 350Lys Gly Val Val Val Asn Ser Ser Val Met Val Lys Phe Leu Phe Pro 355 360365 Arg Pro Asp Gln Gln His Ser Val Ala Tyr Thr Phe Glu Glu Asp Ile 370375 380 Met Thr Thr Val Gln Ala Ser Tyr Ser Lys Lys Lys Leu Phe Leu Ser385 390 395 400 Leu Leu Asp Phe Gln Ile Thr Pro Lys Thr Val Ser Asn LeuThr Glu 405 410 415 Ser Ser Ser Glu Ser Val Gln Ser Phe Leu Gln Ser MetIle Thr Thr 420 425 430 Val Gly Ile Pro Glu Val Met Ser Arg Leu Glu AlaVal Phe Thr Ala 435 440 445 Leu Met Asn Ser Lys Gly Leu Ser Leu Phe AspIle Ile Asn Pro Glu 450 455 460 Ile Ile Thr Arg Asp Gly Phe Leu Leu LeuGln Met Asp Phe Gly Phe 465 470 475 480 Pro Glu His Leu Leu Val Asp PheLeu Gln Ser Leu Ser 485 490 (2) INFORMATION FOR SEQ ID NO:31: (i)SEQUENCE CHARACTERISTICS: (A) LENGTH: 1508 base pairs (B) TYPE: nucleicacid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE:DNA (genomic) (x) PUBLICATION INFORMATION: (A) AUTHORS: Pape, Michael E.Rehberg, Edward F. Marotti, Keith R. Melchior, George W. (B) TITLE:Molecular Cloning, Sequence, and Expression of Cynomolgus MonkeyCholesteryl Ester Transfer Protein (C) JOURNAL: Arteriosclerosis andThrombosis (D) VOLUME: 11 (E) ISSUE: 6 (F) PAGES: 1759-1771 (G) DATE:Nov/Dec-1991 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:31: ATGCTGGCTGCCACCGTCCT GACCCTGGCC CTGCTGGGCA ATGTCCACGC CTGCTCCAAA 60 GGTACCTCACACAAGGCAGG CATTGTGTGC CGCATCACCA AGCCTGCCCT CCTGGTGTTG 120 AACCAACAGACTGCCAAGGT GATCCAGTCT GCCTTCCAGC GAGCCAACTA CCCAAATATC 180 ACAGGCGAGAAGGCCATGAT GCTCCTTGGC CAAGTCAAGT ATGGGTTGCA CAACATCCAA 240 ATCAGCCACTTGTCCATCGC CAGCAGCCGG GTGGAGCTGG TGGAAGCCAA GTCCATTGAT 300 GTCTCCATTCAGAACGTGTC TGTGGTCTTC AAGGGGACCC TGAAGTATGG CTACACCACT 360 GCCTGGGGGCTGGGCATTGA TCAGTCCGTT GACTTCGAGA TCGACTCTGC CATTGACCTC 420 CAGATCAACACACAACTGAC CTGTGACTCT GGTAGAGTGA GGACTGATGC CCCTGACTGC 480 TACCTGTCTTTCCATAAGCT GCTCCTGCAT CTCCAAGGGG AGCGAGAGCC CGGGTGGATC 540 AAGCAGCTGTTCACAAACTT CATCTCCTTC ACCCTGAAGC TGGTCCTGAA GGGACAGATC 600 TGCAAAGAGATCAACATCAT CTCCAACATC ATGGCCGATT TTGTCCAGAC AAGGGCTGCC 660 AGTATCCTTTCAGATGGAGA CATCGGGGTG GACATTTCCC TGACAGGTGA TCCCATCATT 720 ACAGCCTCCTACCTGGAGTC CCATCACAAG GGTTATTTCA TCTATAAGAA TGTCTCGGAG 780 GACCTCCCACTCCCCACCTT CTCGCCCGCA CTGCTGGGGG ACTCCCGCAT GCTGTACTTC 840 TGGTTCTCCGAGCAAGTCTT CCACTCCCTG GCCAAGGTAG CTTTCCAAGA TGCCCGCCTC 900 ACGCTCAGCCTGATGGGAGA CGAGTTCAAG GCAGTGCTGG AGACCTGGGG CTTCAACACC 960 AACCAAGAAATCTTCCAGGA GGTTGTCGGC GGCTTCCCCA GCCAGGCCCA AGTCACCGTC 1020 CACTGCCTCAAGATGCCCAG GATCTCCTGC CAAAACAAGG GAGTCGTGGT CAATTCTTCG 1080 GTGATGGTGAAATTCCTCTT TCCACGCCCA GACCAGCAAC ACTCTGTAGC TTACACATTT 1140 GAAGAGGATATCATGACCAC CGTCCAGGCC TCCTATTCTA AGAAAAAGCT CTTCTTAAGC 1200 CTCTTGGATTTCCAGATTAC ACCAAAGACT GTTTCCAACT TGACTGAGAG CAGCTCCGAG 1260 TCCGTCCAGAGCTTCCTGCA GTCAATGATC ACCACTGTGG GCATCCCTGA GGTCATGTCT 1320 CGGCTTGAGGCAGTGTTTAC AGCCCTCATG AACAGCAAAG GCCTGAGCCT CTTCGACATC 1380 ATCAATCCTGAGATTATCAC TCGAGATGGC TTCCTGCTGC TGCAGATGGA CTTTGGCTTC 1440 CCTGAGCACCTGCTGGTGGA TTTCCTCCAG AGCTTGAGCT AGAAGTCTCC AAGGACGTCA 1500 GGATGGGG1508 (2) INFORMATION FOR SEQ ID NO:32: (i) SEQUENCE CHARACTERISTICS: (A)LENGTH: 20 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: single (D)TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION:SEQ ID NO:32: Gln Glu Ile Phe Gln Glu Val Val Gly Gly Phe Pro Ser GlnAla Gln 1 5 10 15 Val Thr Val His 20 (2) INFORMATION FOR SEQ ID NO:33:(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 20 amino acids (B) TYPE: aminoacid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE:peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO:33: Val Met Val Lys Phe LeuPhe Pro Arg Pro Asp Gln Gln His Ser Val 1 5 10 15 Ala Tyr Thr Phe 20 (2)INFORMATION FOR SEQ ID NO:34: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH:22 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: single (D)TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION:SEQ ID NO:34: Leu Leu Leu Gln Met Asp Phe Gly Phe Pro Glu His Leu LeuVal Asp 1 5 10 15 Phe Leu Gln Ser Leu Ser 20 (2) INFORMATION FOR SEQ IDNO:35: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 20 amino acids (B)TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii)MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO:35: Thr ThrVal Gln Ala Ser Tyr Ser Lys Lys Lys Leu Phe Leu Ser Leu 1 5 10 15 LeuAsp Phe Gln 20 (2) INFORMATION FOR SEQ ID NO:36: (i) SEQUENCECHARACTERISTICS: (A) LENGTH: 20 amino acids (B) TYPE: amino acid (C)STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide(xi) SEQUENCE DESCRIPTION: SEQ ID NO:36: Leu Leu Leu His Leu Gln Gly GluArg Glu Pro Gly Trp Ile Lys Gln 1 5 10 15 Leu Phe Thr Asn 20 (2)INFORMATION FOR SEQ ID NO:37: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH:20 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: single (D)TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION:SEQ ID NO:37: Asn Ile Thr Gly Glu Lys Ala Met Met Leu Leu Gly Gln ValLys Tyr 1 5 10 15 Gly Leu His Asn 20 (2) INFORMATION FOR SEQ ID NO:38:(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 183 amino acids (B) TYPE:amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULETYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:38: Met Asp Ile AspPro Tyr Lys Glu Phe Gly Ala Thr Val Glu Leu Leu 1 5 10 15 Ser Phe LeuPro Ser Asp Phe Phe Pro Ser Val Arg Asp Leu Leu Asp 20 25 30 Thr Ala SerAla Leu Tyr Arg Glu Ala Leu Glu Ser Pro Glu His Cys 35 40 45 Ser Pro HisHis Thr Ala Leu Arg Gln Ala Ile Leu Cys Trp Gly Glu 50 55 60 Leu Met ThrLeu Ala Thr Trp Val Gly Val Asn Leu Glu Asp Pro Ala 65 70 75 80 Ser ArgAsp Leu Val Val Ser Tyr Val Asn Thr Asn Met Gly Leu Lys 85 90 95 Phe ArgGln Leu Leu Trp Phe His Ile Ser Cys Leu Thr Phe Gly Arg 100 105 110 GluThr Val Ile Glu Tyr Leu Val Ser Phe Gly Val Trp Ile Arg Thr 115 120 125Pro Pro Ala Tyr Arg Pro Pro Asn Ala Pro Ile Leu Ser Thr Leu Pro 130 135140 Glu Thr Thr Val Val Arg Arg Arg Gly Arg Ser Pro Arg Arg Arg Thr 145150 155 160 Pro Ser Pro Arg Arg Arg Arg Ser Gln Ser Pro Arg Arg Arg ArgSer 165 170 175 Gln Ser Arg Glu Ser Gln Cys 180 (2) INFORMATION FOR SEQID NO:39: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 552 base pairs (B)TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii)MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:39:ATGGACATCG ACCCTTATAA AGAATTTGGA GCTACTGTGG AGTTACTCTC GTTTTTGCCT 60TCTGACTTCT TTCCTTCAGT ACGAGATCTT CTAGATACCG CCTCAGCTCT GTATCGGGAA 120GCCTTAGAGT CTCCTGAGCA TTGTTCACCT CACCATACTG CACTCAGGCA AGCAATTCTT 180TGCTGGGGGG AACTAATGAC TCTAGCTACC TGGGTGGGTG TTAATTTGGA AGATCCAGCG 240TCTAGAGACC TAGTAGTCAG TTATGTCAAC ACTAATATGG GCCTAAAGTT CAGGCAACTC 300TTGTGGTTTC ACATTTCTTG TCTCACTTTT GGAAGAGAAA CAGTTATAGA GTATTTGGTG 360TCTTTCGGAG TGTGGATTCG CACTCCTCCA GCTTATAGAC CACCAAATGC CCCTATCCTA 420TCAACACTTC CGGAGACTAC TGTTGTTAGA CGACGAGGCA GGTCCCCTAG AAGAAGAACT 480CCCTCGCCTC GCAGACGAAG GTCTCAATCG CCGCGTCGCA GAAGATCTCA ATCTCGGGAA 540TCTCAATGTT AG 552 (2) INFORMATION FOR SEQ ID NO:40: (i) SEQUENCECHARACTERISTICS: (A) LENGTH: 25 amino acids (B) TYPE: amino acid (C)STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide(xi) SEQUENCE DESCRIPTION: SEQ ID NO:40: Thr Trp Val Gly Val Asn Leu GluAsp Pro Ala Ser Arg Asp Leu Val 1 5 10 15 Val Ser Tyr Val Asn Thr AsnMet Gly 20 25 (2) INFORMATION FOR SEQ ID NO:41: (i) SEQUENCECHARACTERISTICS: (A) LENGTH: 21 amino acids (B) TYPE: amino acid (C)STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide(xi) SEQUENCE DESCRIPTION: SEQ ID NO:41: Leu Leu Trp Phe His Ile Ser CysLeu Thr Phe Gly Arg Glu Thr Val 1 5 10 15 Ile Glu Tyr Leu Val 20 (2)INFORMATION FOR SEQ ID NO:42: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH:16 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: single (D)TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION:SEQ ID NO:42: Val Val Ser Tyr Val Asn Thr Asn Met Gly Leu Lys Phe ArgGln Leu 1 5 10 15 (2) INFORMATION FOR SEQ ID NO:43: (i) SEQUENCECHARACTERISTICS: (A) LENGTH: 21 amino acids (B) TYPE: amino acid (C)STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide(xi) SEQUENCE DESCRIPTION: SEQ ID NO:43: Val Ser Phe Gly Val Trp Ile ArgThr Pro Pro Ala Tyr Arg Pro Pro 1 5 10 15 Asn Ala Pro Ile Leu 20 (2)INFORMATION FOR SEQ ID NO:44: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH:35 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D)TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (xi) SEQUENCE DESCRIPTION: SEQID NO:44: GATCCCATGG ACATCGACCC TTATAAAGAA TTTGG 35 (2) INFORMATION FORSEQ ID NO:45: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 44 base pairs(B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear(ii) MOLECULE TYPE: cDNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO:45:GATCAAGCTT TTAACATTGA GATTCCCGAG ATTGAGATCT TCTG 44 (2) INFORMATION FORSEQ ID NO:46: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 43 base pairs(B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear(ii) MOLECULE TYPE: cDNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO:46:GATCGAATTC ACTAGTTGGA AGATCCAGCG TCTAGAGACC TAG 43 (2) INFORMATION FORSEQ ID NO:47: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 41 base pairs(B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear(ii) MOLECULE TYPE: cDNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO:47:GATCGAATTC CTCGAGCTAG AGTCATTAGT TCCCCCCAGC A 41 (2) INFORMATION FOR SEQID NO:48: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 34 base pairs (B)TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii)MOLECULE TYPE: cDNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO:48: GATTATCACTCGAGATGGCT TCCTGCTGCT GCAG 34 (2) INFORMATION FOR SEQ ID NO:49: (i)SEQUENCE CHARACTERISTICS: (A) LENGTH: 40 base pairs (B) TYPE: nucleicacid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE:cDNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO:49: GATCGAATTC AGCGCTCAAGCTCTGGAGGA AATCCACCAG 40 (2) INFORMATION FOR SEQ ID NO:50: (i) SEQUENCECHARACTERISTICS: (A) LENGTH: 26 amino acids (B) TYPE: amino acid (C)STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide(xi) SEQUENCE DESCRIPTION: SEQ ID NO:50: Leu Asp Gly Cys Leu Leu Leu GlnMet Asp Phe Gly Phe Pro Lys His 1 5 10 15 Leu Leu Val Asp Phe Leu GlnSer Leu Ser 20 25 (2) INFORMATION FOR SEQ ID NO:51: (i) SEQUENCECHARACTERISTICS: (A) LENGTH: 51 base pairs (B) TYPE: nucleic acid (C)STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (xi)SEQUENCE DESCRIPTION: SEQ ID NO:51: GGCCGCAACG TTTTACTAGC TCAGGCTCTGCAGGAAATCC ACCAGCAGGT G 51 (2) INFORMATION FOR SEQ ID NO:52: (i)SEQUENCE CHARACTERISTICS: (A) LENGTH: 41 base pairs (B) TYPE: nucleicacid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE:cDNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO:52: GGCCGCCCAT GGCCTGTCCCAAAGGCGCCT CCTACGAGGC T 41

1. A process for producing antibodies to cholesteryl ester transferprotein (CETP) in a mammal that comprises the steps of: (a) immunizingsaid mammal with an inoculum containing a vehicle in which is dissolvedor dispersed a recombinant DNA molecule comprising a DNA sequence thatcontains (i) a sequence encoding a CETP immunogen linked to (ii) apromoter sequence that controls the expression of said CETP immunogenDNA sequence in said mammal, said CETP immunogen being an immunogenicpolypeptide having a CETP amino acid residue sequence, said immunizationproviding an amount of said recombinant DNA molecule sufficient toinduce antibodies to CETP; and (b) maintaining said immunized mammal fora time period sufficient for the production of antibodies that bind toCETP.
 2. The process of claim 1 wherein the blood of said mammalcontains CETP.
 3. A process for increasing the concentration of HDLcholesterol in the blood of a mammal whose blood contains cholesterolester transfer protein (CETP) that comprises the steps of: (a)immunizing said mammal with an inoculum containing a vehicle in which isdissolved or dispersed a recombinant DNA molecule comprising a DNAsequence that contains (i) a sequence encoding a CETP immunogen linkedto (ii) a promoter sequence that controls the expression of said CETPimmunogen DNA sequence in said mammal, said CETP immunogen being animmunogenic polypeptide having a CETP amino acid residue sequence, saidimmunization providing an amount of said recombinant DNA moleculesufficient to induce antibodies to CETP; and (b) maintaining saidimmunized mammal for a time period sufficient for said CETP immunogen tobe expressed and for the production of antibodies that bind to CETP andlessen the transfer of cholesteryl esters from HDL.
 4. The processaccording to claim 3 wherein said immunizing step is repeated.
 5. Theprocess according to claim 3 wherein said immunizing step is repeated atintervals of about 3 to about 6 months until the HDL cholesterol valuein the blood of said mammal is increased by about 10 percent or morerelative to the HDL cholesterol value prior to said first immunizationstep.
 6. The process according to claim 3 wherein said recombinant DNAmolecule encodes human CETP as said immunogenic polypeptide.
 7. Theprocess according to claim 3 wherein said recombinant DNA moleculeencodes rabbit CETP as said immunogenic polypeptide.
 8. The processaccording to claim 3 wherein said encoded CETP immunogen comprises animmunogenic polypeptide fused to an exogenous antigenic carrierpolypeptide.
 9. The process according to claim 8 wherein said exogenousantigenic carrier polypeptide is selected from the group consisting ofhepatitis B core protein, tetanus toxoid, and diphtheria toxoid.
 10. Theprocess according to claim 9 wherein said recombinant DNA moleculeencodes a fusion protein in which said exogenous antigenic carrier isfused to the carboxy-terminus of said immunogenic polypeptide.
 11. Theprocess according to claim 8 wherein the carboxy-terminus of saidencoded exogenous antigenic carrier is fused to the amino-terminus ofsaid encoded immunogenic polypeptide.
 12. The process according to claim8 wherein said encoded exogenous antigenic carrier is fused to both theamino-terminus and carboxy-terminus of said encoded immunogenicpolypeptide.
 13. The process according to claim 12 wherein said encodedfusion protein is comprised of an immunogenic polypeptide having alength of about 10 to about 30 amino acid residues that are fused to anamino-terminal flanking sequence and a carboxy-terminal flankingsequence, wherein (a) said amino-terminal flanking sequence consistsessentially of about 10 to about 20 amino acid residues having an aminoacid residue sequence of the hepatitis B core protein (HBcAg) from aboutposition 1 to about position 35, and said carboxy-terminal sequenceconsists essentially of about 120 to about 160 amino acid residueshaving an amino acid residue sequence of HBcAg from about position 10about position 183, or (b) said amino-terminal flanking sequenceconsists essentially of about 70 to about 90 residues having the aminoacid residue sequence of HBcAg from about position 1 to about position90, and said carboxy-terminal flanking sequence consists essentially ofabout 65 to about 85 amino acid residues having the amino acid residuesequence of HBcAg from about position 80 to about position
 183. 14. Theprocess according to claim 13 wherein the number of amino acid residuespresent in said encoded immunogenic polypeptide is about equal in numberto the number of amino acid residues absent from said HBcAg amino acidresidue sequence between the carboxy-terminal residue position of saidamino-terminal flanking sequence and the amino-terminal residue of saidcarboxy-terminal flanking sequence.
 15. The process according to claim 3wherein said encoded immunogenic polypeptide has the amino acid residuesequence of SEQ ID NOs:29 or
 50. 16. The process according to claim 3wherein said immunization is carried out by injecting said inoculum intomuscle or skin of said mammal.
 17. An inoculum that comprises arecombinant DNA molecule comprising a DNA sequence that contains (i) asequence encoding a CETP immunogen linked to (ii) a promoter sequencethat controls the expression of said CETP immunogen DNA sequence in amammal, said recombinant DNA molecule being dissolved or dispersed in aneffective amount in a vehicle.
 18. The inoculum of claim 17 wherein theconcentration of said DNA encoding said CETP immunogen is about 0.05μg/ml to about 20 mg/ml.
 19. The inoculum of claim 17 wherein saidvehicle is phosphate-buffered saline.
 20. The inoculum of claim 17wherein said vehicle is isotonic sucrose.
 21. The inoculum of claim 17wherein said DNA is complexed with liposomes.